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1
Loewith, Robbie. "A brief history of TOR." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 437–42. http://dx.doi.org/10.1042/bst0390437.
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The TOR (target of rapamycin) serine/threonine kinases are fascinating in that they influence many different aspects of eukaryote physiology including processes often dysregulated in disease. Beginning with the initial characterization of rapamycin as an antifungal agent, studies with yeast have contributed greatly to our understanding of the molecular pathways in which TORs operate. Recently, building on advances in quantitative MS, the rapamycin-dependent phosphoproteome in the budding yeast Saccharomyces cerevisiae was elucidated. These studies emphasize the central importance of TOR and highlight its many previously unrecognized functions. One of these, the regulation of intermediary metabolism, is discussed.
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Wang, Junjun, Xinmiao Ji, Juanjuan Liu, and Xin Zhang. "Serine/Threonine Protein Kinase STK16." International Journal of Molecular Sciences 20, no. 7 (April 10, 2019): 1760. http://dx.doi.org/10.3390/ijms20071760.
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STK16 (Ser/Thr kinase 16, also known as Krct/PKL12/MPSK1/TSF-1) is a myristoylated and palmitoylated Ser/Thr protein kinase that is ubiquitously expressed and conserved among all eukaryotes. STK16 is distantly related to the other kinases and belongs to the NAK kinase family that has an atypical activation loop architecture. As a membrane-associated protein that is primarily localized to the Golgi, STK16 has been shown to participate in the TGF-β signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. This review aims to provide a comprehensive summary of the progress made in recent research about STK16, ranging from its distribution, molecular characterization, post-translational modification (fatty acylation and phosphorylation), interactors (GlcNAcK/DRG1/MAL2/Actin/WDR1), and related functions. As a relatively underexplored kinase, more studies are encouraged to unravel its regulation mechanisms and cellular functions.
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Mukhopadhyay, Subhendu, Vinayak Kapatral, Wenbin Xu, and A. M. Chakrabarty. "Characterization of a Hank’s Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase inPseudomonas aeruginosa." Journal of Bacteriology 181, no. 21 (November 1, 1999): 6615–22. http://dx.doi.org/10.1128/jb.181.21.6615-6622.1999.
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ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screenP. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank’s type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn2+-, but not a Ca2+- or a Mg2+-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.
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Knape, Matthias J., Maximilian Wallbott, Nicole C. G. Burghardt, Daniela Bertinetti, Jan Hornung, Sven H. Schmidt, Robin Lorenz, and Friedrich W. Herberg. "Molecular Basis for Ser/Thr Specificity in PKA Signaling." Cells 9, no. 6 (June 25, 2020): 1548. http://dx.doi.org/10.3390/cells9061548.
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cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.
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Navarro, María N., and Doreen A. Cantrell. "Serine-threonine kinases in TCR signaling." Nature Immunology 15, no. 9 (August 19, 2014): 808–14. http://dx.doi.org/10.1038/ni.2941.
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Sacks, D. B., H. W. Davis, D. L. Crimmins, and J. M. McDonald. "Insulin-stimulated phosphorylation of calmodulin." Biochemical Journal 286, no. 1 (August 15, 1992): 211–16. http://dx.doi.org/10.1042/bj2860211.
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Calmodulin is phosphorylated in vitro by the insulin-receptor tyrosine kinase and a variety of serine/threonine kinases. Here we report that insulin stimulates the phosphorylation of calmodulin on average 3-fold in intact rat hepatocytes. Although calmodulin is constitutively phosphorylated, insulin increases phosphate incorporation into serine, threonine and tyrosine residues. We demonstrate that casein kinase II, an insulin-sensitive kinase, phosphorylates calmodulin in vitro on serine/thyronine residues (Thr-79, Ser-81, Ser-101 and Thr-117). The ability of the insulin receptor to phosphorylate calmodulin that has been pre-phosphorylated by casein kinase II is enhanced up to 35-fold, and the sites of phosphorylation on calmodulin are shifted from tyrosine to threonine and serine. These observations, obtained with a new specific monoclonal antibody to calmodulin, confirm that insulin stimulates calmodulin phosphorylation in intact cells. The observation that calmodulin is phosphorylated in vivo, coupled with the recent demonstration that phosphocalmodulin exhibits altered biological activity, strongly suggests that phosphorylation of calmodulin is a critical component of intracellular signalling.
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BAJPAI, Anil, and Zacharie BRAHMI. "Regulation of natural killer cell-mediated cytotoxicity by serine/threonine phosphatases: identification of a calyculin A-sensitive serine/threonine kinase." Biochemical Journal 320, no. 1 (November 15, 1996): 153–59. http://dx.doi.org/10.1042/bj3200153.
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We have recently reported that Ser/Thr phosphatases play a key role in regulating natural killer (NK) cell lytic activity and that calyculin A and okadaic acid affect this activity differently [Bajpai and Brahmi (1994) J. Biol. Chem. 269, 18864–18869]. Here, we investigate a mechanism that might account for this differential action of calyculin A and okadaic acid on NK cells. Calyculin A specifically inhibited the lytic activity of YT-INDY, an NK-like cell line, and hyperphosphorylated 60 and 78 kDa proteins. The kinetics of appearance of these two proteins was correlated with the loss of lytic activity. In contrast, okadaic acid did not significantly affect either of these activities. The 78 kDa protein is localized in the cytosolic compartment whereas the 60 kDa protein is distributed equally between the membrane and the cytosolic fractions. Both proteins display a kinase activity and are phosphorylated mainly at serine and threonine residues but not at tyrosine residues. The activation of these kinases is specific to calyculin A treatment; it is independent of protein kinase C, protein kinase A, Ca2+, phosphotyrosine phosphatase and protein synthesis de novo. In conclusion, we have demonstrated that calyculin A, but not okadaic acid, hyperphosphorylates two proteins with Ser/Thr kinase activity, thus explaining the differential regulation of NK cells by these two Ser/Thr phosphatase inhibitors.
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Nádvorník, Richard, Tomáš Vomastek, Jiří Janeček, Zuzana Techniková, and Pavel Branny. "Pkg2, a Novel Transmembrane Protein Ser/Thr Kinase of Streptomyces granaticolor." Journal of Bacteriology 181, no. 1 (January 1, 1999): 15–23. http://dx.doi.org/10.1128/jb.181.1.15-23.1999.
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ABSTRACT A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed inEscherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.
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Av-Gay, Yossef, Sarwat Jamil, and Steven J. Drews. "Expression and Characterization of the Mycobacterium tuberculosis Serine/Threonine Protein Kinase PknB." Infection and Immunity 67, no. 11 (November 1, 1999): 5676–82. http://dx.doi.org/10.1128/iai.67.11.5676-5682.1999.
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ABSTRACT PknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. The pknBgene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis ofpknB expression in Mycobacterium tuberculosisindicates the presence of pknB mRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis.
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Songyang, Z., K. P. Lu, Y. T. Kwon, L. H. Tsai, O. Filhol, C. Cochet, D. A. Brickey, et al. "A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1." Molecular and Cellular Biology 16, no. 11 (November 1996): 6486–93. http://dx.doi.org/10.1128/mcb.16.11.6486.
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We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.
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Verma, Anita, and Anthony T. Maurelli. "Identification of Two Eukaryote-Like Serine/Threonine Kinases Encoded by Chlamydia trachomatis Serovar L2 and Characterization of Interacting Partners of Pkn1." Infection and Immunity 71, no. 10 (October 2003): 5772–84. http://dx.doi.org/10.1128/iai.71.10.5772-5784.2003.
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ABSTRACT Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.
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Hoekstra, M. F., N. Dhillon, G. Carmel, A. J. DeMaggio, R. A. Lindberg, T. Hunter, and J. Kuret. "Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity." Molecular Biology of the Cell 5, no. 8 (August 1994): 877–86. http://dx.doi.org/10.1091/mbc.5.8.877.
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We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.
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PHALIP, Vincent, Jian-Hong LI, and Cheng-Cai ZHANG. "HstK, a cyanobacterial protein with both a serine/threonine kinase domain and a histidine kinase domain: implication for the mechanism of signal transduction." Biochemical Journal 360, no. 3 (December 10, 2001): 639–44. http://dx.doi.org/10.1042/bj3600639.
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Two distinct families of protein kinases are involved in signal transduction: Ser, Thr and Tyr kinases, which are predominantly found among eukaryotes, and His kinases, as part of bacterial two-component signalling systems. Genetic studies in Arabidopsis and Saccharomyces have demonstrated that bacterial-type two-component systems may act upstream of Ser/Thr kinases in the same signalling pathway, but how this coupling is accomplished remains unclear. In the present study, we report the characterization of a protein kinase, HstK, from the N2-fixing cyanobacterium Anabaena sp. PCC 7120, that possesses both a Ser/Thr kinase domain and a His kinase domain. Proteins with a structural architecture similar to that of HstK can be found in the eukaryote, Schizosaccharomyces pombe, and the bacterium, Rhodococcus sp. M5. HstK was present in cells grown with NH4+ or N2 as the nitrogen source, but was absent in cells grown with NO3−. The hstK gene was inactivated and the mutant phenotype was characterized. The catalytic domain of the Ser/Thr kinase of HstK functionally replaced that of Hog1p, a well-characterized protein kinase required for the response to high osmolarity in the S. cerevisiae heterologous system. The unusual multidomain structure of HstK suggests that a two-component system could be directly coupled to Ser/Thr kinases in the same signal transduction pathway.
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Rossomando, A. J., P. Dent, T. W. Sturgill, and D. R. Marshak. "Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation." Molecular and Cellular Biology 14, no. 3 (March 1994): 1594–602. http://dx.doi.org/10.1128/mcb.14.3.1594.
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Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.
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Rossomando, A. J., P. Dent, T. W. Sturgill, and D. R. Marshak. "Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation." Molecular and Cellular Biology 14, no. 3 (March 1994): 1594–602. http://dx.doi.org/10.1128/mcb.14.3.1594-1602.1994.
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Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.
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Lasserre, Rémi, Céline Cuche, Ronnie Blecher-Gonen, Evgeny Libman, Elise Biquand, Anne Danckaert, Deborah Yablonski, Andrés Alcover, and Vincenzo Di Bartolo. "Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation." Journal of Cell Biology 195, no. 5 (November 21, 2011): 839–53. http://dx.doi.org/10.1083/jcb.201103105.
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Antigen recognition within immunological synapses triggers and sustains T cell activation by nucleating protein microclusters that gather T cell receptors (TCRs), kinases, and adaptors. Dissipation of these microclusters results in signal termination, but how this process is regulated is unclear. In this paper, we reveal that release of the adaptors SLP76 and GADS from signaling microclusters is induced by the serine/threonine protein kinase HPK1 and that phosphorylation of GADS plays a major role in this process. We found that HPK1 was recruited into microclusters and triggered their dissipation by inducing the phosphorylation of a threonine-containing motif of GADS, together with the previously described serine phosphorylation of SLP76. These events induced the cooperative binding of 14-3-3 proteins to SLP76–GADS complexes, leading to their uncoupling from the transmembrane adaptor LAT and consequently reducing microcluster persistence and activation-induced gene transcription. These results demonstrate that serine/threonine phosphorylation of multiple TCR-proximal effectors controls the stability of signaling microclusters, thereby determining the intensity of T cell responses.
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Jang, Jichan, Alexandre Stella, Frédéric Boudou, Florence Levillain, Eliette Darthuy, Julien Vaubourgeix, Chongzhen Wang, et al. "Functional characterization of the Mycobacterium tuberculosis serine/threonine kinase PknJ." Microbiology 156, no. 6 (June 1, 2010): 1619–31. http://dx.doi.org/10.1099/mic.0.038133-0.
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Eukaryotic-like Ser/Thr protein kinases (STPKs) are present in many bacterial species, where they control various physiological and virulence processes by enabling microbial adaptation to specific environmental signals. PknJ is the only member of the 11 STPKs identified in Mycobacterium tuberculosis that still awaits characterization. Here we report that PknJ is a functional kinase that forms dimers in vitro, and contains a single transmembrane domain. Using a high-density peptide-chip-based technology, multiple potential mycobacterial targets were identified for PknJ. We confirmed PknJ-dependent phosphorylation of four of these targets: PknJ itself, which autophosphorylates at Thr168, Thr171 and Thr173 residues; the transcriptional regulator EmbR; the methyltransferase MmaA4/Hma involved in mycolic acid biosynthesis; and the dipeptidase PepE, whose encoding gene is located next to pknJ in the mycobacterial genome. Our results provide a number of candidate phospho-targets for PknJ and possibly other mycobacterial STPKs that could be studied to investigate the role of STPKs in M. tuberculosis physiology and virulence.
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PHIN, Sopheap, Deborah KUPFERWASSER, Joseph LAM, and Kay K. LEE-FRUMAN. "Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1." Biochemical Journal 373, no. 2 (July 15, 2003): 583–91. http://dx.doi.org/10.1042/bj20021794.
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Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.
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Kornbluth, S., B. Sebastian, T. Hunter, and J. Newport. "Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14." Molecular Biology of the Cell 5, no. 3 (March 1994): 273–82. http://dx.doi.org/10.1091/mbc.5.3.273.
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The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.
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Lu, Xiangyi, Lizabeth A. Perkins, and Norbert Perrimon. "The torso pathway in Drosophila: a model system to study receptor tyrosine kinase signal transduction." Development 119, Supplement (December 1, 1993): 47–56. http://dx.doi.org/10.1242/dev.119.supplement.47.
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In the Drosophila embryo, specification of terminal cell fates that result in the formation of both the head (acron) and tail (telson) regions is under the control of the torso (tor) receptor tyrosine kinase. The current knowledge suggests that activation of tor at the egg pole initiates a signal transduction pathway that is mediated sequentially by the guanine nucleotide releasing factor son of sevenless (Sos), the p21Ras1 GTPase, the serine/threonine kinase D-raf and the tyrosine/threonine kinase MAPKK (Dsor1). Subsequently, it is postulated that activation, possibly by phosphorylation, of a transcription factor at the egg poles activates the transcription of the terminal gap genes tailless and huckebein. These gap genes, which encode putative transcription factors, then control the expression of more downstream factors that ultimately result in head and tail differentiation. Also involved in tor signaling is the non-receptor protein tyrosine phosphatase corkscrew (csw). Here, we review the current model and discuss future research directions in this field.
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KATZ, Sigal, and Ami ARONHEIM. "Differential targeting of the stress mitogen-activated protein kinases to the c-Jun dimerization protein 2." Biochemical Journal 368, no. 3 (December 15, 2002): 939–45. http://dx.doi.org/10.1042/bj20021127.
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The mitogen-activated kinases are structurally related proline-directed serine/threonine kinases that phosphorylate similar phosphoacceptor sites and yet, in vivo, they exhibit stringent substrate specificity. Specific targeting domains (kinase docking domains) facilitate kinase—substrate interaction and play a major role in substrate specificity determination. The c-Jun N-terminal kinase (JNK) consensus docking domain comprises of a KXXK/RXXXXLXL motif located in the Δ-domain of the c-Jun N-terminal to the phosphoacceptor site. The c-Jun dimerization protein 2 is phosphorylated by JNK on Thr-148. Activating transcription factor 3 (ATF3) is a basic leucine zipper protein which is highly homologous to c-Jun dimerization protein 2 (JDP2), especially within the threonine/proline phosphoacceptor site, Thr-148. Nevertheless, ATF3 does not serve as a JNK substrate in vitro or in vivo. Using ATF3 and JDP2 protein chimaeras, we mapped the JNK-docking domain within JDP2. Although a JNK consensus putative docking site is located within the JDP2 leucine zipper motif, this domain does not function to recruit JNK to JDP2. A novel putative docking domain located C-terminally to the JDP2 phosphoacceptor site was identified. This domain, when fused to the ATF3 heterologous phosphoacceptor site, can direct its phosphorylation by JNK. In addition, although the novel JNK-docking domain was found to be necessary for p38 phosphorylation of JDP2 on Thr-148, it was not sufficient to confer JDP2 phosphorylation by the p38 kinase.
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Chen, R. H., C. Sarnecki, and J. Blenis. "Nuclear localization and regulation of erk- and rsk-encoded protein kinases." Molecular and Cellular Biology 12, no. 3 (March 1992): 915–27. http://dx.doi.org/10.1128/mcb.12.3.915.
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We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus.
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Chen, R. H., C. Sarnecki, and J. Blenis. "Nuclear localization and regulation of erk- and rsk-encoded protein kinases." Molecular and Cellular Biology 12, no. 3 (March 1992): 915–27. http://dx.doi.org/10.1128/mcb.12.3.915-927.1992.
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We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus.
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Blume, Constanze, Peter M. Benz, Ulrich Walter, Joohun Ha, Bruce E. Kemp, and Thomas Renné. "AMP-activated Protein Kinase Impairs Endothelial Actin Cytoskeleton Assembly by Phosphorylating Vasodilator-stimulated Phosphoprotein." Journal of Biological Chemistry 282, no. 7 (November 2, 2006): 4601–12. http://dx.doi.org/10.1074/jbc.m608866200.
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Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that links signaling pathways to remodeling of the cytoskeleton. VASP functions are modulated by protein kinases, which phosphorylate the sites Ser-157, Ser-239, and Thr-278. The kinase responsible for Thr-278 phosphorylation, biological functions of the phosphorylation, and association with disease states have remained enigmatic. Using VASP phosphorylation status-specific antibodies, we identified AMP-activated protein kinase (AMPK), a serine-threonine kinase and fundamental sensor of energy homeostasis, in a screen for kinases that phosphorylate the Thr-278 site of VASP in endothelial cells. Pharmacological AMPK inhibitors and activators and AMPK mutants revealed that the kinase specifically targets residue Thr-278 but not Ser-157 or Ser-239. Quantitative fluorescence-activated cell sorter analysis and serum response factor transcriptional reporter assays, which quantify the cellular F-/G-actin equilibrium, indicated that AMPK-mediated VASP phosphorylation impaired actin stress fiber formation and altered cell morphology. In the Zucker Diabetic Fatty (ZDF) rat model for type II diabetes, AMPK activity and Thr-278 phosphorylation were substantially reduced in arterial vessel walls. These findings suggest that VASP is a new AMPK substrate, that VASP Thr-278 phosphorylation translates metabolic signals into actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessels.
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Izquierdo, M., S. Bowden, and D. Cantrell. "The role of Raf-1 in the regulation of extracellular signal-regulated kinase 2 by the T cell antigen receptor." Journal of Experimental Medicine 180, no. 1 (July 1, 1994): 401–6. http://dx.doi.org/10.1084/jem.180.1.401.
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Triggering of the T cell antigen receptor (TCR) complex activates the serine/threonine kinase Raf-1 whose function is necessary for TCR induction of the interleukin 2 gene. Raf-1 has been identified as a candidate mitogen-activated protein (MAP) kinase kinase kinase (MKKK) and thus has the potential to couple the TCR to the activation of the MAP kinases such as ERK2. In the present study, the role of Raf-1 in ERK2 regulation of ERK2 in T cells has been explored. A constitutively active Raf-1 kinase, v-raf, or a dominant inhibitory Raf-1 mutant were expressed transiently from the pEF BOS vector in Jurkat cells and the effects of these Raf-1 mutants on a coexpressed ERK2 reporter was assessed. The action of the constitutively active Raf-1 was to stimulate the ERK2 kinase, whereas the dominant negative version of Raf-1 inhibited the ERK2 activation induced by triggering of the TCR. These data indicate a role for Raf-1 in the regulation of ERK2 in T cells.
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Kanellopoulou, Chryssa, Alex B. George, Evan Masutani, Jennifer L. Cannons, Juan C. Ravell, Tori N. Yamamoto, Margery G. Smelkinson, et al. "Mg2+ regulation of kinase signaling and immune function." Journal of Experimental Medicine 216, no. 8 (June 13, 2019): 1828–42. http://dx.doi.org/10.1084/jem.20181970.
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Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2–inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.
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Mugume, Yosia, Zakayo Kazibwe, and Diane C. Bassham. "Target of Rapamycin in Control of Autophagy: Puppet Master and Signal Integrator." International Journal of Molecular Sciences 21, no. 21 (November 4, 2020): 8259. http://dx.doi.org/10.3390/ijms21218259.
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The target of rapamycin (TOR) is an evolutionarily-conserved serine/threonine kinase that senses and integrates signals from the environment to coordinate developmental and metabolic processes. TOR senses nutrients, hormones, metabolites, and stress signals to promote cell and organ growth when conditions are favorable. However, TOR is inhibited when conditions are unfavorable, promoting catabolic processes such as autophagy. Autophagy is a macromolecular degradation pathway by which cells degrade and recycle cytoplasmic materials. TOR negatively regulates autophagy through phosphorylation of ATG13, preventing activation of the autophagy-initiating ATG1-ATG13 kinase complex. Here we review TOR complex composition and function in photosynthetic and non-photosynthetic organisms. We also review recent developments in the identification of upstream TOR activators and downstream effectors of TOR. Finally, we discuss recent developments in our understanding of the regulation of autophagy by TOR in photosynthetic organisms.
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Suzuki, Toru, Satoru Matsuda, Junko K. Tsuzuku, Yutaka Yoshida, and Tadashi Yamamoto. "A serine/threonine kinase p90rsk1 phosphorylates the anti-proliferative protein Tob." Genes to Cells 6, no. 2 (February 2001): 131–38. http://dx.doi.org/10.1046/j.1365-2443.2001.00406.x.
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Kato, Akihisa, Jun Arii, Ikuo Shiratori, Hiroomi Akashi, Hisashi Arase, and Yasushi Kawaguchi. "Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral Envelope Glycoprotein B and Regulates Its Expression on the Cell Surface." Journal of Virology 83, no. 1 (October 22, 2008): 250–61. http://dx.doi.org/10.1128/jvi.01451-08.
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ABSTRACT Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). As reported here, we attempted to identify the previously unreported physiological substrate of Us3 in HSV-1-infected cells. Our results were as follows. (i) Bioinformatics analysis predicted two putative Us3 phosphorylation sites in the viral envelope glycoprotein B (gB) at codons 557 to 562 (RRVSAR) and codons 884 to 889 (RRNTNY). (ii) In in vitro kinase assays, the threonine residue at position 887 (Thr-887) in the gB domain was specifically phosphorylated by Us3, while the serine residue at position 560 was not. (iii) The phosphorylation of gB Thr-887 in Vero cells infected with wild-type HSV-1 was specifically detected using an antibody that recognized phosphorylated serine or threonine residues with arginine at the −3 and −2 positions. (iv) The phosphorylation of gB Thr-887 in infected cells was dependent on the kinase activity of Us3. (v) The replacement of Thr-887 with alanine markedly upregulated the cell surface expression of gB in infected cells, whereas replacement with aspartic acid, which sometimes mimics constitutive phosphorylation, restored the wild-type phenotype. The upregulation of gB expression on the cell surface also was observed in cells infected with a recombinant HSV-1 encoding catalytically inactive Us3. These results supported the hypothesis that Us3 phosphorylates gB and downregulates the cell surface expression of gB in HSV-1-infected cells.
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Miron, Mathieu, Paul Lasko, and Nahum Sonenberg. "Signaling from Akt to FRAP/TOR Targets both 4E-BP andS6K in Drosophilamelanogaster." Molecular and Cellular Biology 23, no. 24 (December 15, 2003): 9117–26. http://dx.doi.org/10.1128/mcb.23.24.9117-9126.2003.
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ABSTRACT The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted us to examine the regulation of d4E-BP in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. We used RNA interference (RNAi) to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affected insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation.
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Gelperin, Daniel, Lynn Horton, Anne DeChant, Jack Hensold, and Sandra K. Lemmon. "Loss of Ypk1 Function Causes Rapamycin Sensitivity, Inhibition of Translation Initiation and Synthetic Lethality in 14-3-3-Deficient Yeast." Genetics 161, no. 4 (August 1, 2002): 1453–64. http://dx.doi.org/10.1093/genetics/161.4.1453.
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Abstract 14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR.
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Fujinaga, Koh, Thomas P. Cujec, Junmin Peng, Judit Garriga, David H. Price, Xavier Graña, and B. Matija Peterlin. "The Ability of Positive Transcription Elongation Factor b To Transactivate Human Immunodeficiency Virus Transcription Depends on a Functional Kinase Domain, Cyclin T1, and Tat." Journal of Virology 72, no. 9 (September 1, 1998): 7154–59. http://dx.doi.org/10.1128/jvi.72.9.7154-7159.1998.
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ABSTRACT By binding to the transactivation response element (TAR) RNA, the transcriptional transactivator (Tat) from the human immunodeficiency virus increases rates of elongation rather than initiation of viral transcription. Two cyclin-dependent serine/threonine kinases, CDK7 and CDK9, which phosphorylate the C-terminal domain of RNA polymerase II, have been implicated in Tat transactivation in vivo and in vitro. In this report, we demonstrate that CDK9, which is the kinase component of the positive transcription elongation factor b (P-TEFb) complex, can activate viral transcription when tethered to the heterologous Rev response element RNA via the regulator of expression of virion proteins (Rev). The kinase activity of CDK9 and cyclin T1 is essential for these effects. Moreover, P-TEFb binds to TAR only in the presence of Tat. We conclude that Tat–P-TEFb complexes bind to TAR, where CDK9 modifies RNA polymerase II for the efficient copying of the viral genome.
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Morrison, Donna L., Jasbinder S. Sanghera, Justine Stewart, Steven L. Pelech, Cindy Sutherland, and Michael P. Walsh. "Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1." Biochemistry and Cell Biology 74, no. 4 (July 1, 1996): 549–57. http://dx.doi.org/10.1139/o96-459.
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Smooth muscle myosin light chain kinase (MLCK) features several consensus sites of phosphorylation by proline-directed protein serine/threonine kinases. The phosphorylation of MLCK by two proline-directed kinases isolated from sea star oocytes, i.e., p44mpk (Mpk, a mitogen-activated protein kinase homologue) and cyclin-dependent kinase-1 (CDK1, also known as p34cdc2), was investigated. Chicken gizzard MLCK was phosphorylated on seryl and fhreonyl residues by both Mpk and CDK1. Phosphorylation of MLCK to 0.6 mol Pi/mol by Mpk increased the Vmax of phosphotransferase activity towards a synthetic peptide corresponding to residues 11–23 of the 20-kDa light chain of myosin by 1.6-fold. Phosphorylation of MLCK to 1.0 mol Pi/mol by CDK1 increased the Vmax by 2.3-fold. Phosphorylation by either kinase had no significant effect on the concentration of calmodulin required for half-maximal activation of MLCK. Analysis of the phosphorylation of synthetic peptides containing consensus phosphorylation sites for Mpk and CDK1 indicated that the major site of phosphorylation in MLCK by Mpk was Ser-834, and by CDK1 was Thr-283. Both of these sites are located outside the cafmodulin-binding site (residues 796–815), consistent with the observation that phosphorylation by Mpk or CDK1 was unaffected by the presence of bound Ca2+/calmodulin. These results indicate that MLCK activity may be regulated by phosphorylation catalyzed by proline-directed kinases, possibly directed at Thr-40 and Thr-43 at the amino terminus of MLCK.Key words: myosin light chain kinase, mitogen-activated protein kinase, cyclin-dependent kinase.
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Ye, Junjie, Mingjun Shi, Wei Chen, Feng Zhu, and Qiuhong Duan. "Research Advances in the Molecular Functions and Relevant Diseases of TAOKs, Novel STE20 Kinase Family Members." Current Pharmaceutical Design 26, no. 26 (August 11, 2020): 3122–33. http://dx.doi.org/10.2174/1381612826666200203115458.
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As serine/threonine-protein kinases, Thousand and One Kinases(TAOKs) are members of the GCKlike superfamily, one of two well-known branches of the Ste20 kinase family. Within the last two decades, three functionally similar kinases, namely TAOK1-3, were identified. TAOKs are involved in many molecular and cellular events. Scholars widely believe that TAOKs act as kinases upstream of the MAPK cascade and as factors that interact with MST family kinases, the cytoskeleton, and apoptosis-associated proteins. Therefore, TAOKs are thought to function in tumorigenesis. Additionally, TAOKs participate in signal transduction induced by Notch, TCR, and IL-17. Recent studies found that TAOKs play roles in a series of diseases and conditions, such as the central nervous system dysfunction, herpes viral infection, immune system imbalance, urogenital system malformation during development, cardiovascular events, and childhood obesity. Therefore, inhibitory chemicals targeting TAOKs may be of great significance as potential drugs for these diseases.
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Tyler, Jessica S., and David I. Friedman. "Characterization of a Eukaryotic-Like Tyrosine Protein Kinase Expressed by the Shiga Toxin-Encoding Bacteriophage 933W." Journal of Bacteriology 186, no. 11 (June 1, 2004): 3472–79. http://dx.doi.org/10.1128/jb.186.11.3472-3479.2004.
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ABSTRACT The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.
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Wang, Hong, and David L. Brautigan. "A Novel Transmembrane Ser/Thr Kinase Complexes with Protein Phosphatase-1 and Inhibitor-2." Journal of Biological Chemistry 277, no. 51 (October 21, 2002): 49605–12. http://dx.doi.org/10.1074/jbc.m209335200.
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Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr320, which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required aVTFmotif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.
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Stokoe, D., B. Caudwell, P. T. W. Cohen, and P. Cohen. "The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2." Biochemical Journal 296, no. 3 (December 15, 1993): 843–49. http://dx.doi.org/10.1042/bj2960843.
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The substrate specificity of mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MAPKAP kinase-2) was investigated by using synthetic peptides related to the N-terminus of glycogen synthase. The minimum sequence required for efficient phosphorylation was found to be Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (Phe > Leu > Val >> Ala), and the peptide Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala was phosphorylated with a Km of 9.3 microM and Vmax. of 10 mumol/min per mg. MAPKAP kinase-1 (a homologue of ribosomal protein S6 kinase) also requires an arginine three residues N-terminal to the serine (position n-3), but not a hydrophobic residue at position n-5. Neither MAPKAP kinase-1 nor MAPKAP kinase-2 could tolerate a proline residue at position n + 1, indicating that their specificities do not overlap with that of MAP kinase. The specificity of calmodulin-dependent protein kinase-II resembled that of MAPKAP kinase-2, except that it could tolerate replacement of the arginine by a lysine and the phosphorylation-site serine by a threonine residue. Partial cDNAs encoding MAPKAP kinase-2 were isolated from rabbit and human skeletal muscle and human teratocarcinoma libraries, and Northern-blotting experiments revealed a single 3.3 kb mRNA transcript present at similar levels in six human tissues examined. The catalytic domain was most similar (35-40% identity) to calmodulin-dependent protein kinases II and IV, phosphorylase kinase, putative serine kinase H1 and the C-terminal domain of MAPKAP kinase-1, which form one branch of the protein kinase phylogenetic tree. The sequence N-terminal to the catalytic domain is proline-rich and contains two putative SH3-binding sites. The threonine residue phosphorylated by MAP kinase lies immediately C-terminal to the catalytic domain and is followed by a nuclear localization signal, Lys-Lys-(Xaa)10-Lys-Arg-Arg-Lys-Lys, near the C-terminus.
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Tafur, Lucas, Jennifer Kefauver, and Robbie Loewith. "Structural Insights into TOR Signaling." Genes 11, no. 8 (August 4, 2020): 885. http://dx.doi.org/10.3390/genes11080885.
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The Target of Rapamycin (TOR) is a highly conserved serine/threonine protein kinase that performs essential roles in the control of cellular growth and metabolism. TOR acts in two distinct multiprotein complexes, TORC1 and TORC2 (mTORC1 and mTORC2 in humans), which maintain different aspects of cellular homeostasis and orchestrate the cellular responses to diverse environmental challenges. Interest in understanding TOR signaling is further motivated by observations that link aberrant TOR signaling to a variety of diseases, ranging from epilepsy to cancer. In the last few years, driven in large part by recent advances in cryo-electron microscopy, there has been an explosion of available structures of (m)TORC1 and its regulators, as well as several (m)TORC2 structures, derived from both yeast and mammals. In this review, we highlight and summarize the main findings from these reports and discuss both the fascinating and unexpected molecular biology revealed and how this knowledge will potentially contribute to new therapeutic strategies to manipulate signaling through these clinically relevant pathways.
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Kang, Junmei, and Zhen Wang. "Mut9p-LIKE KINASE Family Members: New Roles of the Plant-Specific Casein Kinase I in Plant Growth and Development." International Journal of Molecular Sciences 21, no. 5 (February 25, 2020): 1562. http://dx.doi.org/10.3390/ijms21051562.
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Casein kinase I (CK1), a ubiquitous serine/threonine (Ser/Thr) protein kinase in eukaryotes, plays pivotal roles in a wide spectrum of cellular functions including metabolism, cell cycle progression, developmental control and stress response. Plant CK1 evolves a lineage expansion, resulting in a unique branch of members exclusive to the kingdom. Among them, Arabidopsis Mut9p-LIKE KINASEs (MLKs) target diverse substrates including histones and the key regulatory proteins involving in physiological processes of light signaling, circadian rhythms, phytohormone and plant defense. Deregulation of the kinase activity by mutating the enzyme or the phosphorylation sites of substrates causes developmental disorders and susceptibility to adverse environmental conditions. Recent findings suggest that MLKs have evolved as a general kinase that modifies transcription factors or primary regulatory proteins in a dynamic way. Here, we summarize the current knowledge of the roles of MLKs and MLK orthologs in several commercially important crops.
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Murakami, Mirei, Tomoko Ichisaka, Mitsuyo Maeda, Noriko Oshiro, Kenta Hara, Frank Edenhofer, Hiroshi Kiyama, Kazuyoshi Yonezawa, and Shinya Yamanaka. "mTOR Is Essential for Growth and Proliferation in Early Mouse Embryos and Embryonic Stem Cells." Molecular and Cellular Biology 24, no. 15 (August 1, 2004): 6710–18. http://dx.doi.org/10.1128/mcb.24.15.6710-6718.2004.
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ABSTRACT TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells.
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Klevernic, Iva V., Margaret J. Stafford, Nicholas Morrice, Mark Peggie, Simon Morton, and Philip Cohen. "Characterization of the reversible phosphorylation and activation of ERK8." Biochemical Journal 394, no. 1 (January 27, 2006): 365–73. http://dx.doi.org/10.1042/bj20051288.
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ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15–20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.
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Wang, Bin, Shifan Yang, Lei Zhang, and Zheng-Guo He. "Archaeal Eukaryote-Like Serine/Threonine Protein Kinase Interacts with and Phosphorylates a Forkhead-Associated-Domain-Containing Protein." Journal of Bacteriology 192, no. 7 (January 29, 2010): 1956–64. http://dx.doi.org/10.1128/jb.01471-09.
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ABSTRACT Protein phosphorylation plays an important role in cell signaling. However, in the Archaea, little is known about which proteins are phosphorylated and which kinases are involved. In this study, we identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase and its protein partner, a forkhead-associated (FHA)-domain-containing protein, from the archaeon Sulfolobus tokodaii strain 7. This protein kinase, ST1565, physically interacted with the FHA-domain-containing protein, ST0829, both in vivo and in vitro. ST1565 preferred Mn2+ as a cofactor for autophosphorylation and for substrate phosphorylation; the optimal temperature for this was 45°C, and the optimal pH was 5.5 to 7.5. The critical amino acid residues of the conserved FHA and kinase domain sites were identified by performing a series of mutation assays. Thr329 was part of a major activation site in the kinase, while Thr326 was a negative regulation site. Several mutants with amino acid substitutions in the conserved FHA domain sites of ST0829 did not physically interact with ST1565. A structural model for the FHA domain demonstrated that the mutation sites were located at the edge of the protein and thus were in the domain that potentially interacts with ST1565. This report describes pioneering work on the third domain of life, the Archaea, showing that a protein kinase interacts with and phosphorylates an FHA-domain-containing protein. Our data provide critical information on the structural or functional characteristics of archaeal proteins and could help increase our understanding of fundamental signaling mechanisms in all three domains of life.
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Brunet, Anne, Jongsun Park, Hien Tran, Linda S. Hu, Brian A. Hemmings, and Michael E. Greenberg. "Protein Kinase SGK Mediates Survival Signals by Phosphorylating the Forkhead Transcription Factor FKHRL1 (FOXO3a)." Molecular and Cellular Biology 21, no. 3 (February 1, 2001): 952–65. http://dx.doi.org/10.1128/mcb.21.3.952-965.2001.
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ABSTRACT Serum- and glucocorticoid-inducible kinases (SGKs) form a novel family of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. SGKs are related to Akt (also called PKB), a serine/threonine kinase that plays a crucial role in promoting cell survival. Like Akt, SGKs are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation. However the physiological substrates and cellular functions of SGKs remained to be identified. We hypothesized that SGKs regulate cellular functions in concert with Akt by phosphorylating common targets within the nucleus. The best-characterized nuclear substrates of Akt are transcription factors of the Forkhead family. Akt phosphorylates Forkhead transcription factors such as FKHRL1, leading to FKHRL1's exit from the nucleus and the consequent shutoff of FKHRL1 target genes. We show here that SGK1, like Akt, promotes cell survival and that it does so in part by phosphorylating and inactivating FKHRL1. However, SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1. While both kinases can phosphorylate Thr-32, SGK displays a marked preference for Ser-315 whereas Akt favors Ser-253. These findings suggest that SGK and Akt may coordinately regulate the function of FKHRL1 by phosphorylating this transcription factor at distinct sites. The efficient phosphorylation of these three sites on FKHRL1 by SGK and Akt appears to be critical to the ability of growth factors to suppress FKHRL1-dependent transcription, thereby preventing FKHRL1 from inducing cell cycle arrest and apoptosis. These findings indicate that SGK acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression.
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Charest, D. L., G. Mordret, K. W. Harder, F. Jirik, and S. L. Pelech. "Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1." Molecular and Cellular Biology 13, no. 8 (August 1993): 4679–90. http://dx.doi.org/10.1128/mcb.13.8.4679.
Full textAbstract:
p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
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Charest, D. L., G. Mordret, K. W. Harder, F. Jirik, and S. L. Pelech. "Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1." Molecular and Cellular Biology 13, no. 8 (August 1993): 4679–90. http://dx.doi.org/10.1128/mcb.13.8.4679-4690.1993.
Full textAbstract:
p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
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Paquette, Mathieu, Leeanna El-Houjeiri, and Arnim Pause. "mTOR Pathways in Cancer and Autophagy." Cancers 10, no. 1 (January 12, 2018): 18. http://dx.doi.org/10.3390/cancers10010018.
Full textAbstract:
TOR (target of rapamycin), an evolutionarily-conserved serine/threonine kinase, acts as a central regulator of cell growth, proliferation and survival in response to nutritional status, growth factor, and stress signals. It plays a crucial role in coordinating the balance between cell growth and cell death, depending on cellular conditions and needs. As such, TOR has been identified as a key modulator of autophagy for more than a decade, and several deregulations of this pathway have been implicated in a variety of pathological disorders, including cancer. At the molecular level, autophagy regulates several survival or death signaling pathways that may decide the fate of cancer cells; however, the relationship between autophagy pathways and cancer are still nascent. In this review, we discuss the recent cellular signaling pathways regulated by TOR, their interconnections to autophagy, and the clinical implications of TOR inhibitors in cancer.
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Ruiz-Medina, Blanca E., Jeremy A. Ross, and Robert A. Kirken. "Interleukin-2 Receptor β Thr-450 Phosphorylation Is a Positive Regulator for Receptor Complex Stability and Activation of Signaling Molecules." Journal of Biological Chemistry 290, no. 34 (July 7, 2015): 20972–83. http://dx.doi.org/10.1074/jbc.m115.660654.
Full textAbstract:
T, B, and natural killer cells are required for normal immune response and are regulated by cytokines such as IL-2. These cell signals are propagated following receptor-ligand engagement, controlling recruitment and activation of effector proteins. The IL-2 receptor β subunit (IL-2Rβ) serves in this capacity and is known to be phosphorylated. Tyrosine phosphorylation of the β chain has been studied extensively. However, the identification and putative regulatory roles for serine and threonine phosphorylation sites have yet to be fully characterized. Using LC-MS/MS and phosphospecific antibodies, a novel IL-2/IL-15 inducible IL-2Rβ phosphorylation site (Thr-450) was identified. IL-2 phosphokinetic analysis revealed that phosphorylation of IL-2Rβ Thr-450 is rapid (2.5 min), transient (peaks at 15 min), and protracted compared with receptor tyrosine phosphorylation and occurs in multiple cell types, including primary human lymphocytes. Pharmacological and siRNA-mediated inhibition of various serine/threonine kinases revealed ERK1/2 as a positive regulator, whereas purified protein phosphatase 1 (PP1), dephosphorylated Thr-450 in vitro. Reconstitution assays demonstrated that Thr-450 is important for regulating IL-2R complex formation, recruitment of JAK3, and activation of AKT and ERK1/2 and a transcriptionally active STAT5. These results provide the first evidence of the identification and functional characterization for threonine phosphorylation of an interleukin receptor.
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Otsubo, Yoko, Yoshiaki Kamada, and Akira Yamashita. "Novel Links between TORC1 and Traditional Non-Coding RNA, tRNA." Genes 11, no. 9 (August 19, 2020): 956. http://dx.doi.org/10.3390/genes11090956.
Full textAbstract:
Target of rapamycin (TOR) is a serine/threonine kinase that modulates cell growth and metabolism in response to environmental changes. Transfer RNA (tRNA) is an abundant and ubiquitous small non-coding RNA that is essential in the translation of mRNAs. Beyond its canonical role, it has been revealed that tRNAs have more diverse functions. TOR complex 1 (TORC1), which is one of the two TOR complexes, regulates tRNA synthesis by controlling RNA polymerase III. In addition to tRNA synthesis regulation, recent studies have revealed hidden connections between TORC1 and tRNA, which are both essential players in eukaryotic cellular activities. Here, we review the accumulating findings on the regulatory links between TORC1 and tRNA—particularly those links in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe.
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Taagepera, S., P. Dent, J. H. Her, T. W. Sturgill, and G. J. Gorbsky. "The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183." Molecular Biology of the Cell 5, no. 11 (November 1994): 1243–51. http://dx.doi.org/10.1091/mbc.5.11.1243.
Full textAbstract:
Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition.
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Chang, Yu-Yun, Gábor Juhász, Pankuri Goraksha-Hicks, Andrew M. Arsham, Daniel R. Mallin, Laura K. Muller, and Thomas P. Neufeld. "Nutrient-dependent regulation of autophagy through the target of rapamycin pathway." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 232–36. http://dx.doi.org/10.1042/bst0370232.
Full textAbstract:
In response to nutrient deficiency, eukaryotic cells activate macroautophagy, a degradative process in which proteins, organelles and cytoplasm are engulfed within unique vesicles called autophagosomes. Fusion of these vesicles with the endolysosomal compartment leads to breakdown of the sequestered material into amino acids and other simple molecules, which can be used as nutrient sources during periods of starvation. This process is driven by a group of autophagy-related (Atg) proteins, and is suppressed by TOR (target of rapamycin) signalling under favourable conditions. Several distinct kinase complexes have been implicated in autophagic signalling downstream of TOR. In yeast, TOR is known to control autophagosome formation in part through a multiprotein complex containing the serine/threonine protein kinase Atg1. Recent work in Drosophila and mammalian systems suggests that this complex and its regulation by TOR are conserved in higher eukaryotes, and that Atg1 has accrued additional functions including feedback regulation of TOR itself. TOR and Atg1 also control the activity of a second kinase complex containing Atg6/Beclin 1, Vps (vacuolar protein sorting) 15 and the class III PI3K (phosphoinositide 3-kinase) Vps34. During autophagy induction, Vps34 activity is mobilized from an early endosomal compartment to nascent autophagic membranes, in a TOR- and Atg1-responsive manner. Finally, the well-known TOR substrate S6K (p70 ribosomal protein S6 kinase) has been shown to play a positive role in autophagy, which may serve to limit levels of autophagy under conditions of continuously low TOR activity. Further insight into these TOR-dependent control mechanisms may support development of autophagy-based therapies for a number of pathological conditions.