Journal articles: 'PLEKHA1 [Pleckstrin homology domain containing'

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Rouaud, Florian, Francesca Tessaro, Laura Aimaretti, Leonardo Scapozza, and Sandra Citi. "Cooperative binding of the tandem WW domains of PLEKHA7 to PDZD11 promotes conformation-dependent interaction with tetraspanin 33." Journal of Biological Chemistry 295, no. 28 (May 5, 2020): 9299–312. http://dx.doi.org/10.1074/jbc.ra120.012987.

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Pleckstrin homology domain–containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain–containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin–mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S-transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a “hydrophobic hot spot” region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7–PDZD11 interaction modulate the ligand-binding properties of PLEKHA7.

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Yayici Köken, Özlem, Ülkühan Öztoprak, Vehap Topçu, Büsranur Çavdarli, Çagri Mesut Temucin, Üstün Aydingöz, Özge Dedeoglu Toptas, Hulya Kayilioglu, and Deniz Yuksel. "Expanding the genotype-phenotype spectrum of autosomal recessive Charcot-Marie-Tooth disease: A novel PLEKHG5 gene mutation." Neurology Asia 26, no. 3 (September 2021): 607–12. http://dx.doi.org/10.54029/2021jmr.

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Autosomal recessive intermediate Charcot Marie Tooth (CMT) disease type C is a very rarely-seen neurogenetic disorder. Homozygous or compound heterozygous mutation in the Pleckstrin homology domain-containing family G member 5 (PLEKHG5) gene on chromosome 1p36 was recently reported in patients with CMT. From the first description of the disease to date, almost 40 different variants associated with the PLEKHG5 gene were identified. Here, we present an adolescent girl who was thought initially to be myopathy because of progressive proximal muscle weakness. The electrophysiologic study revealed axonal sensory and motor neuropathy with some demyelinating features. She was diagnosed with autosomal recessive inheritance, intermediate CMT disease type C with a novel homozygous mutation in the PLEKHG5 gene in clinical exome sequencing as c.1600- 2A>G by next-generation sequencing. We describe here the novel mutation in the PLEKHG5 gene and the genotype-phenotype correlation.

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Xing, Xiangling, Ninni Mu, Xiaotian Yuan, Na Wang, C. Christofer Juhlin, Klas Strååt, Catharina Larsson, and Dawei Xu. "PLEKHS1 Over-Expression is Associated with Metastases and Poor Outcomes in Papillary Thyroid Carcinoma." Cancers 12, no. 8 (July 31, 2020): 2133. http://dx.doi.org/10.3390/cancers12082133.

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Pleckstrin homology domain containing S1 (PLEKHS1) is a poorly characterized factor, although its promoter mutations were identified in human malignancies including thyroid carcinoma (TC). This study was designed to determine PLEKHS1 promoter hotspot mutations in papillary and anaplastic thyroid carcinomas (PTCs and ATCs) and to evaluate if PLEKHS1 expression influences clinical outcome. The PLEKHS1 promoter mutation was observed in 1/93 of PTCs and none of 18 ATCs in our cohort; however, PLEKHS1 expression was aberrantly up-regulated in TCs compared to adjacent non-tumorous thyroid tissues. ATC tumors, an undifferentiated TC, exhibited the highest PLEKHS1 expression. In both TCGA and present cohorts of PTCs, PLEKHS1 gene methylation density was inversely correlated with its mRNA expression and demethylation at the PLEKHS1 locus occurred at two CpGs. Higher PLEKHS1 expression was associated with lymph node and distant metastases, and shorter overall and disease-free survival in our cohort of PTC patients. Importantly, PLEKHS1 over-expression predicted shorter patient survival in PTCs lacking TERT promoter mutations. Cellular experiments showed that PLEKHS1 over-expression enhanced AKT phosphorylation and invasiveness. Collectively, the PLEKHS1 gene demethylation causes its over-expression in PTCs. PLEKHS1 promotes aggressive behavior of TCs possibly by increasing AKT activity, and its over-expression predicts poor patient outcomes.

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Spellmann, Ilja, Dan Rujescu, Richard Musil, Ina Giegling, Just Genius, Peter Zill, Sandra Dehning, et al. "Pleckstrin homology domain containing 6 protein (PLEKHA6) polymorphisms are associated with psychopathology and response to treatment in schizophrenic patients." Progress in Neuro-Psychopharmacology and Biological Psychiatry 51 (June 2014): 190–95. http://dx.doi.org/10.1016/j.pnpbp.2014.02.006.

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Reyes, H. D., E. Devor, A. M. Newtson, Y. A. Lyons, M. McDonald, V. M. Wagner, J. N. Mattson, K. K. Leslie, and J. Gonzalez-Bosquet. "Expression of Pleckstrin Homology and RUN Domain Containing M1 (PLEKHM1) is significantly associated with Grade and Prognosis in Endometrial Adenocarcinoma." Gynecologic Oncology 156, no. 3 (March 2020): e18. http://dx.doi.org/10.1016/j.ygyno.2019.11.071.

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Nguyen, Trang Thi Thu, Wei Sun Park, Byung Ouk Park, Cha Yeon Kim, Yohan Oh, Jin Man Kim, Hana Choi, et al. "PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front." Proceedings of the National Academy of Sciences 113, no. 36 (August 23, 2016): 10091–96. http://dx.doi.org/10.1073/pnas.1604720113.

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Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.

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AshaRani, P. V., Syidda Amron, Noor Azizah Bte Zainuldin, Sumanty Tohari, Alvin Y. J. Ng, Guo Song, Byrappa Venkatesh, and Ajay S. Mathuru. "Whole-Exome Sequencing to Identify Potential Genetic Risk in Substance Use Disorders: A Pilot Feasibility Study." Journal of Clinical Medicine 10, no. 13 (June 25, 2021): 2810. http://dx.doi.org/10.3390/jcm10132810.

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Genetics intersects with environmental, cultural, and social factors in the development of addictive disorders. This study reports the feasibility of whole-exome sequencing of trios (subject and two family members) to discover potential genetic variants in the development of substance use disorders (SUD). Family trios were recruited from the National Addictions Management Service in Singapore during the 2016–2018 period. Recruited subjects had severe alcohol use disorder (AUD) or opioid use disorder (OUD), with nicotine dependence (ND) and a family history of addictive disorders. Demographic characteristics and severity of addiction were captured. Whole-exome sequencing (WES) and analysis were performed on salivary samples collected from the trios. WES revealed variants in several genes in each individual and disruptive protein mutations in most. Variants were identified in genes previously associated with SUDs, such as Pleckstrin homology domain-containing family M member 3 (PLEKHM3), coiled-coil serine-rich protein 1 (CCSER1), LIM and calponin homology domains-containing protein 1 (LIMCH1), dynein axonemal heavy chain 8 (DNAH8), and the taste receptor type 2 member 38 (TAS2R38) involved in the perception of bitterness. The feasibility study suggests that subjects with a severe addiction profile, polysubstance use, and family history of addiction may often harbor gene variants that may predispose them to SUDs. This study could serve as a model for future precision medicine-based personalized interventional strategies for behavioral addictions and SUDs and for the discovery of potentially pathogenic genetic variants.

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Tran, Thuy T., Chetan K. Rane, Christopher R. Zito, Sarah A. Weiss, Shlomit Jessel, Liliana Lucca, Benjamin Y. Lu, et al. "Clinical Significance of PDCD4 in Melanoma by Subcellular Expression and in Tumor-Associated Immune Cells." Cancers 13, no. 5 (March 2, 2021): 1049. http://dx.doi.org/10.3390/cancers13051049.

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Little is known about the subcellular localization and function of programmed cell death 4 (PDCD4) in melanoma. Our past studies suggest PDCD4 interacts with Pleckstrin Homology Domain Containing A5 (PLEKHA5) to influence melanoma brain metastasis outcomes, as high intracranial PDCD4 expression leads to improved survival. We aimed to define the subcellular distribution of PDCD4 in melanoma and in the tumor microenvironment during neoplastic progression and its impact on clinical outcomes. We analyzed multiple tissue microarrays with well-annotated clinicopathological variables using quantitative immunofluorescence and evaluated single-cell RNA-sequencing on a brain metastasis sample to characterize PDCD4+ immune cell subsets. We demonstrate differences in PDCD4 expression during neoplastic progression, with high tumor and stromal PDCD4 levels associated with improved survival in primary melanomas and in intracranial metastases, but not in extracranial metastatic disease. While the expression of PDCD4 is well-documented on CD8+ T cells and natural killer cells, we show that it is also found on B cells and mast cells. PDCD4 expression in the tumor microenvironment is associated with increased immune cell infiltration. Further studies are needed to define the interaction of PDCD4 and PLEKHA5 and to evaluate the utility of this pathway as a therapeutic target in melanoma brain metastasis.

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Tellermann, A., T. Witte, C. Lansche, M. Stoll, RE Schmidt, and NT Baerlecken. "Autoantibodies binding to ubiquitin-fold modifier-conjugating enzyme 1 (Ufc1) and pleckstrin homology domain containing, family G (with RhoGef domain) member 2 (Plekhg2) are associated with mycobacterial infections." HIV Medicine 16, no. 2 (September 12, 2014): 114–21. http://dx.doi.org/10.1111/hiv.12194.

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Spellmann, Ilja, Dan Rujescu, Richard Musil, Sebastian Meyer, Ina Giegling, Just Genius, Peter Zill, et al. "Corrigendum to “Pleckstrin homology domain containing 6 protein (PLEKHA6) polymorphisms are associated with psychopathology and response to treatment in schizophrenic patients” [Prog Neuro-Psychopharmacol Biol Psychiatry 51 (2014) 190–195]." Progress in Neuro-Psychopharmacology and Biological Psychiatry 58 (April 2015): 106. http://dx.doi.org/10.1016/j.pnpbp.2014.11.007.

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Nowak, Daniel, Norihiko Kawamata, Birte Niebuhr, Verena Nowak, Maximilian Mossner, Rahul R. Nahar, Nils Heinrich Thoennissen, et al. "The Pax5 Fusion Product Pax5-C20orf112 Causes Downregulation of Pre-B Cell Receptor Genes and Induces Differential Proliferation Patterns in B-Lymphoblastic Cell Lines." Blood 114, no. 22 (November 20, 2009): 1284. http://dx.doi.org/10.1182/blood.v114.22.1284.1284.

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Abstract Abstract 1284 Poster Board I-306 Recent SNP array analyses of B-acute lymphoblastic leukemia (B-ALL) have identified disruptions of the gene encoding the B-cell specific transcription factor Pax5 as one of the most common genomic lesions in this disease (> 30%); it being hemizygously deleted, mutated or involved in translocations. Pax5 translocates and forms fusion products with at least 12 different partners including C20orf112, leading to a chimeric Pax5/C20orf112 (Pax5/C20s) protein. Pax5 fusion products act as dominant negatives, competing for promoter binding sites with wild type (wt) Pax5 and thereby deregulating expression of target genes. In order to elucidate the molecular effects of fusion products involving the Pax5 gene, we performed a global gene expression analysis in the Nalm-6 B-ALL cell line. The cells were transfected with MSCV expression plasmids containing either empty vector, wild type Pax5 or a short fusion product of Pax5 and C20orf112 (Pax5/C20s), each containing IRES sequences for co-expression of GFP. Overexpression of Pax5 and Pax5/C20s was confirmed by western blot and quantitative RT PCR. RNA was extracted from cells sorted by FACS for GFP and processed for hybridization on Affymetrix HG-U133 plus 2 gene expression microarrays. Candidate genes were validated with RT real time PCR. Among the most differentially downregulated genes by the Pax5/C20s fusion product were candidate genes such as pleckstrin homology domain containing, family A member 2 (PLEKHA2) (12.64-fold), B-cell associated transcription factors POU class 2 associating factor 1 (POU2AF1) (4.4-fold) and transcription factor 3 (TCF3, E2A) (3.9-fold). Another intriguing observation was the downregulation of a group of genes associated with signaling through the pre-B cell receptor such as phosphoinositide-3-kinase adaptor protein 1 (BCAP) (3.35 fold), immunoglobulin heavy locus (IGH) (2.8 fold), pre-B lymphocyte genes -3 and -1 (VPREB3, VPREB1) (2.6-fold and 1.75-fold, respectively), spleen tyrosine kinase (SYK) (1.6 fold) and B-cell linker (SLP65, BLNK) (1.5-fold) by the Pax5/C20s fusion product. For stable expression and growth curves, Nalm6, 697, Kasumi2, RCH-ACV, SEM, HPB-Null, BV173 and BEL1 B-lymphoblastic cell lines were infected with retroviruses expressing the above mentioned retroviral expression constructs. We noted that forced expression of the PAX5/C20s fusion product inhibited growth in cell lines, which had functional pre-B cell receptor signaling. In contrast, the fusion gene either did not affect or enhanced growth of B-ALL cell lines, in which expression of a functional pre-B cell receptor was missing. Of note, Pax5 wt caused growth inhibition in B-ALL cell lines lacking functional pre-B cell receptor signaling. In cells with functional pre-B cell signaling, the response to engagement of the receptor as measured by calcium flux assay was diminished by overexpression of the Pax5/C20s fusion product as compared to empty vector control or PAX5 wt. These results suggest that the mechanisms of leukemogenesis of Pax5/C20s in ALL cells may be dependent on the functionality of the pre-B cell receptor pathway. This could be of great therapeutic value as it would potentially allow ALL cells to be divided into two different subtypes depending on pre-B cell receptor functionality and possibly identify the pre-B cell receptor pathway as a new therapeutic target. Disclosures No relevant conflicts of interest to declare.

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Ren, Xiu-Rong, Quan-Sheng Du, Yang-Zhong Huang, Shi-Zhou Ao, Lin Mei, and Wen-Cheng Xiong. "Regulation of Cdc42 Gtpase by Proline-Rich Tyrosine Kinase 2 Interacting with Psgap, a Novel Pleckstrin Homology and Src Homology 3 Domain Containing Rhogap Protein." Journal of Cell Biology 152, no. 5 (March 5, 2001): 971–84. http://dx.doi.org/10.1083/jcb.152.5.971.

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Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain–dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

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Bach, Tami L., Wesley T. Kerr, Yanfeng Wang, Eve Marie Bauman, Purnima Kine, Eileen L. Whiteman, Renell S. Morgan, et al. "PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization." Blood 109, no. 3 (September 28, 2006): 1147–55. http://dx.doi.org/10.1182/blood-2006-02-001339.

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Abstract Pleckstrin-2 is composed of 2 pleckstrin homology (PH) domains and a disheveled–Egl-10–pleckstrin (DEP) domain. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to D3 and D5 phosphoinositides. Pleckstrin-2 expressed in Jurkat T cells bound to the cellular membrane and enhanced actin-dependent spreading only after stimulation of the T-cell antigen receptor or the integrin α4β1. A pleckstrin-2 variant containing point mutations in both PH domains failed to associate with the Jurkat membrane and had no effect on spreading under the same conditions. Although still membrane bound, a pleckstrin-2 variant containing point mutations in the DEP domain demonstrated a decreased ability to induce membrane ruffles and spread. Pleckstrin-2 also colocalized with actin at the immune synapse and integrin clusters via its PH domains. Although pleckstrin-2 can bind to purified D3 and D5 phosphoinositides, the intracellular membrane association of pleckstrin-2 and cell spreading are dependent on D3 phosphoinositides, because these effects were disrupted by pharmacologic inhibition of phosphatidylinositol 3-kinase (PI3K). Our results indicate that pleckstrin-2 uses its modular domains to bind to membrane-associated phosphatidylinositols generated by PI3K, whereby it coordinates with the actin cytoskeleton in lymphocyte spreading and immune synapse formation.

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Kohn, Aimee D., Fumito Takeuchi, and Richard A. Roth. "Akt, a Pleckstrin Homology Domain Containing Kinase, Is Activated Primarily by Phosphorylation." Journal of Biological Chemistry 271, no. 36 (September 6, 1996): 21920–26. http://dx.doi.org/10.1074/jbc.271.36.21920.

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DOWLER, Simon, Richard A. CURRIE, David G. CAMPBELL, Maria DEAK, Gursant KULAR, C. Peter DOWNES, and Dario R. ALESSI. "Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities." Biochemical Journal 351, no. 1 (October 1, 2000): 19. http://dx.doi.org/10.1042/0264-6021:3510019.

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DOWLER, Simon, Richard A. CURRIE, David G. CAMPBELL, Maria DEAK, Gursant KULAR, C. Peter DOWNES, and Dario R. ALESSI. "Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities." Biochemical Journal 351, no. 1 (September 26, 2000): 19–31. http://dx.doi.org/10.1042/bj3510019.

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The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P3. The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P3. Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P3, but instead possess unexpected and novel phosphoinositide-binding specificitiesin vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P2 [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P2 (centaurin-β2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s).

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de Bruyn, Kim M. T., Johan de Rooij, Rob M. F. Wolthuis, Holger Rehmann, Joep Wesenbeek, Robbert H. Cool, Alfred H. Wittinghofer, and Johannes L. Bos. "RalGEF2, a Pleckstrin Homology Domain Containing Guanine Nucleotide Exchange Factor for Ral." Journal of Biological Chemistry 275, no. 38 (July 10, 2000): 29761–66. http://dx.doi.org/10.1074/jbc.m001160200.

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Ma, Alice, and Charles Abrams. "Pleckstrin Homology Domains and Phospholipid-Induced Cytoskeletal Reorganization." Thrombosis and Haemostasis 82, no. 08 (1999): 399–406. http://dx.doi.org/10.1055/s-0037-1615859.

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IntroductionA remarkable event that takes place during platelet activation is the reorganization that occurs when platelets adhere and spread on exposed collagen fibrils or become activated in the circulation by agonists, such as thrombin or adenosine diphosphate (ADP). In response to either stimulus, the shape of the platelet changes from a smooth disc to an irregular form with multiple, finger-like projections. This transformation is due to cytoskeletal rearrangements within the platelet. The platelet cytoskeleton is an intricately woven network1 arranged in three major structures: a cytoplasmic actin network, a rim of membrane-associated cytoskeleton, and a marginal band consisting of a microtubule coil. Together, these lend support to the platelet plasma membrane and give shape to both resting and activated platelets.At several levels, phosphoinositides are involved in the regulation of the platelet cytoskeleton. Actin binding, capping, and severing proteins are regulated by binding to phosphatidylinositol 4,5-bisphosphate (PIP2). The action of specific phosphoinositide kinases and phosphatases, leading to the regulation of levels of D3- and D4-containing phosphoinositides, has a profound impact on actin assembly. For example, synthesis of D3-containing phosphoinositides by phosphoinositide 3-kinases (PI3Ks) can lead to cortical actin assembly and the formation of lamellipodia downstream of stimulation by growth factor receptors, insulin receptors, and G protein-coupled receptors.2-5 There is increasing evidence that other lipid kinases also regulate cytoskeletal reorganization. Phosphatidylinositol 4-P 5-kinase enzymes, acting downstream of Rho family GTPases, have been shown to stimulate actin assembly.6 Because these areas have been covered in other articles,7,8 this review will, instead, concentrate on the role of pleckstrin and pleckstrin homology (PH) domains in the regulation of the actin cytoskeleton.Pleckstrin homology (PH) domains are the most wellrecognized phosphoinositide-binding protein motifs, comprising “modules” within more than 100 signaling proteins, and are used to mediate intermolecular interactions. The threedimensional structures of all PH domains studied to date are virtually superimposable, despite divergence in their amino acid sequence.9-17 The basic PH domain structure is composed of a β “sandwich,” capped at one end by a carboxyl-terminal α-helix, and all PH domains exhibit a striking polarity in their distribution of surface charge (Fig. 1). Based on the similarity of the structure of the NH2-terminal PH domain of pleckstrin to that of the retinol-binding protein, which was known to bind lipids, Harlan and coworkers tested PH domains and demonstrated that they bind to phosphoinositides.18 Since then, a number of laboratories, including our own, have published reports showing that the binding of PH domains to phosphoinositides can regulate protein function.4,19-22 It is now accepted that PH domains serve to localize their molecules into membrane structures by binding to phosphoinositides;18,23 though some PH domains may interact with other targets, such as the βγ subunits of heterotrimeric G proteins (Gβγ)24-27 or protein kinase C (PKC).28-30 The structure of several PH domains complexed to inositol trisphosphate (IP3) has been solved,11,13 confirming a physical interaction between the inositol phosphate headgroup and the positively charged face of the PH domain. For example, the association of the PH domain of PLCδ with IP3 is shown in Figure 1. Pleckstrin is a 43-kDa hematopoietic protein that contains the amino- and carboxyl- termini of the two prototypic PH domains (Fig. 2). Pleckstrin was first described as a major substrate for PKC in platelets and leukocytes, and its phosphorylation has long been used as a marker for platelet activation. Though its function in vivo remains unclear, expressed pleckstrin can affect PIP2-based signaling mediated by phospholipase C, PI3K, and inositol phosphatases.31-33 Ser113, Thr114, and Ser117, the three residues phosphorylated by PKC, lie adjacent to, but not within, the amino-terminal PH domain. Phosphorylation at these sites has been shown to regulate the function of this PH domain.34 Recently, a third functional motif has been described within pleckstrin.35 This motif is termed the DEP domain after the first three proteins known to possess this sequence (disheveled, Egl-10, and pleckstrin).

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Berg, J. S., B. H. Derfler, C. M. Pennisi, D. P. Corey, and R. E. Cheney. "Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin." Journal of Cell Science 113, no. 19 (October 1, 2000): 3439–51. http://dx.doi.org/10.1242/jcs.113.19.3439.

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Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with <45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil. Like several other unconventional myosins and a plant kinesin, myosin-X contains both a myosin tail homology 4 (MyTH4) domain and a FERM (band 4.1/ezrin/radixin/moesin) domain. The unique tail domain also includes three pleckstrin homology domains, which have been implicated in phosphatidylinositol phospholipid signaling, and three PEST sites, which may allow cleavage of the myosin tail. Most intriguingly, myosin-X in cultured cells is present at the edges of lamellipodia, membrane ruffles, and the tips of filopodial actin bundles. The tail domain structure, biochemical features, and localization of myosin-X suggest that this novel unconventional myosin plays a role in regions of dynamic actin.

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XU, Yue, Li-Fong SEET, Brendon HANSON, and Wanjin HONG. "The Phox homology (PX) domain, a new player in phosphoinositide signalling." Biochemical Journal 360, no. 3 (December 10, 2001): 513–30. http://dx.doi.org/10.1042/bj3600513.

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Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P2, it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.

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Flesch, Frits M., Jong W. Yu, Mark A. Lemmon, and Koert N. J. Burger. "Membrane activity of the phospholipase C-δ1 pleckstrin homology (PH) domain." Biochemical Journal 389, no. 2 (July 5, 2005): 435–41. http://dx.doi.org/10.1042/bj20041721.

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PH-PLCδ1 [the PH domain (pleckstrin homology domain) of PLCδ1 (phospholipase C-δ1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCδ1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCδ1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCδ1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCδ1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCδ1 cannot. PH-PLCδ1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCδ1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCδ1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain.

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Bach, Tami L., Wesley T. Kerr, and Charles S. Abrams. "PI3K Regulates Pleckstrin-2 in T-Cell Cytoskeletal Re-Organization." Blood 106, no. 11 (November 16, 2005): 3305. http://dx.doi.org/10.1182/blood.v106.11.3305.3305.

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Abstract Pleckstrin-2, a paralog of pleckstrin-1, is composed of two Pleckstrin Homology (PH) domains and a Disheveled-Egl 10-Pleckstrin (DEP) domain. Several studies have shown that PH domains mediate binding of their host proteins to inositol phosphates and phospholipids, and thus regulate protein function. PH domains are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. Proteins containing the DEP domain also regulate a broad range of cellular functions and evidence is emerging that several signaling proteins may rely on their DEP domains for membrane association. We speculated that the function of pleckstrin-2 is dependent upon its ability to bind to specific polyphosphatidylinositols. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to the products of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bind to the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. To examine the effect of pleckstrin-2 in human T-cells, Jurkat T-cells were transfected with GFP-tagged plasmids that direct the expression of pleckstrin-2 variants. Using confocal video microscopy we demonstrated that upon activation of the T-cell antigen receptor or the integrin α4β1, pleckstrin-2 rapidly moves from the cytoplasm to the cellular membrane and enhances membrane ruffling. Quantitation of cell footprint size revealed a two-fold increase in cell spreading. Furthermore, the membrane association of pleckstrin-2 and its resultant cell spreading were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. Consistent with this observation, a pleckstrin-2 variant containing point mutations in both of its PH domains failed to associate with the cell membrane and had no effect on spreading under the same conditions, suggesting that pleckstrin-2 membrane association occurs through a pathway dependent on the phospholipid-binding pocket of its PH domains. Although still membrane-bound, a pleckstin-2 variant containing point mutations in the second β-turn and second α-helical coil of the DEP domain demonstrated a decreased ability of pleckstrin-2 to induce membrane ruffles and lamellipodia, without decreasing filopodia formation. The cell footprint size of the DEP domain mutants was also decreased compared to that of wild type pleckstrin-2. These results suggest that the pleckstrin-2 DEP domain may function to promote actin-rich membrane extensions and ruffling. The localization of receptors, signaling intermediates, and cytoskeletal components at the T-cell/APC interface is thought to be a major determinant of efficient T-cell activation. Our data indicate that in T-lymphocytes, pleckstrin-2 uses modular motifs to bind to membrane-associated phosphatidylinositols, such as those generated by PI3K, to organize the actin cytoskeleton and to promote lymphocyte spreading.

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Gorai, Sukhamoy, Debasish Paul, Nandan Haloi, Rituparna Borah, Manas Kumar Santra, and Debasis Manna. "Mechanistic insights into the phosphatidylinositol binding properties of the pleckstrin homology domain of lamellipodin." Molecular BioSystems 12, no. 3 (2016): 747–57. http://dx.doi.org/10.1039/c5mb00731c.

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24

Ni, Tao, Antreas C. Kalli, Fiona B. Naughton, Luke A. Yates, Omar Naneh, Mirijam Kozorog, Gregor Anderluh, Mark S. P. Sansom, and Robert J. C. Gilbert. "Structure and lipid-binding properties of the kindlin-3 pleckstrin homology domain." Biochemical Journal 474, no. 4 (February 3, 2017): 539–56. http://dx.doi.org/10.1042/bcj20160791.

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Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain (4.1-erythrin–radixin–moiesin domain) comprising F0–F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at a resolution of 2.23 Å and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP)-binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1–β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared with PtdIns(4,5)P2. Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as an analyte indicate affinities of 300 µM for PtdIns(3,4,5)P3 and 1 mM for PtdIns(4,5)P2. In contrast, SPR studies with an immobilised PH domain and lipid nanodiscs as the analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (∼5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1–β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance of lipid mobility and clustering for the biophysical assessment of protein–membrane interactions.

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Yu, Wen-Mei, Teresa S. Hawley, Robert G. Hawley, and Cheng-Kui Qu. "Role of the docking protein Gab2 in β1-integrin signaling pathway-mediated hematopoietic cell adhesion and migration." Blood 99, no. 7 (April 1, 2002): 2351–59. http://dx.doi.org/10.1182/blood.v99.7.2351.

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Gab2, a newly identified pleckstrin homology domain-containing docking protein, is a major binding protein of SHP-2 tyrosine phosphatase in interleukin (IL)-3–stimulated hematopoietic cells. Its signaling mechanism remains largely unknown. We report here an important regulatory role for Gab2 in β1 integrin signaling pathway that mediates hematopoietic cell adhesion and migration. Cross-linking of the β1 integrin on Ba/F3 cells induced rapid tyrosine phosphorylation of Gab2 and its association with Syk kinase, SHP-2 phosphatase, and the p85 subunit of phosphatidylinositol (PI)-3 kinase. In addition, Gab2 was also constitutively associated with SHP-1 phosphatase via its C-terminal Src homology 2 domain. Overexpression of the pleckstrin homology domain or a mutant Gab2 molecule lacking SHP-2 binding sites resulted in significant reductions in Ba/F3 cell adhesion and migration. Biochemical analyses revealed that enforced expression of Gab2 mutant molecules dramatically reduced β1-integrin ligation-triggered PI3 kinase activation, whereas Erk kinase activation remained unaltered. Furthermore, transduction of primary hematopoietic progenitor cells from viable motheaten mice with these mutant Gab2 molecules also significantly ameliorated their enhanced migration capacity associated with theSHP1 gene mutation. Taken together, these results suggest an important signaling role for Gab2 in regulating hematopoietic cell adhesion and migration.

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Xu, Shunbin, Yanshu Wang, Haiqing Zhao, Lilei Zhang, Weihong Xiong, King-Wai Yau, Hakim Hiel, Elisabeth Glowatzki, David K. Ryugo, and David Valle. "PHR1, a PH Domain-Containing Protein Expressed in Primary Sensory Neurons." Molecular and Cellular Biology 24, no. 20 (October 15, 2004): 9137–51. http://dx.doi.org/10.1128/mcb.24.20.9137-9151.2004.

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ABSTRACT Previously, we identified PHR1 as an abundantly expressed gene in photoreceptors and showed that it encodes four isoforms, each with N-terminal pleckstrin homology (PH) and C-terminal transmembrane domains. To better understand PHR1 function and expression, we made a Phr1 null mouse by inserting a β-galactosidase/neor cassette into exon 3. In addition to photoreceptors, we found abundant expression of specific Phr1 splice forms in olfactory receptor neurons and vestibular and cochlear hair cells. We also found Phr1 expression in cells with a possible sensory function, including peripheral retinal ganglion cells, cochlear interdental cells, and neurons of the circumventricular organ. Despite this discrete expression in known and putative sensory neurons, mice lacking PHR1 do not have overt sensory deficits.

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Del Fattore, Andrea, Rachele Fornari, Liesbeth Van Wesenbeeck, Fenna de Freitas, Jean-Pierre Timmermans, Barbara Peruzzi, Alfredo Cappariello, et al. "A New Heterozygous Mutation (R714C) of the Osteopetrosis Gene, Pleckstrin Homolog Domain Containing Family M (With Run Domain) Member 1 (PLEKHM1), Impairs Vesicular Acidification and Increases TRACP Secretion in Osteoclasts." Journal of Bone and Mineral Research 23, no. 3 (November 12, 2007): 380–91. http://dx.doi.org/10.1359/jbmr.071107.

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28

Roll, Richard L., Eve Marie Bauman, Joel S. Bennett, and Charles S. Abrams. "Phosphorylated Pleckstrin Induces Cell Spreading via an Integrin-Dependent Pathway." Journal of Cell Biology 150, no. 6 (September 18, 2000): 1461–66. http://dx.doi.org/10.1083/jcb.150.6.1461.

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Pleckstrin is a 40-kD phosphoprotein containing NH2- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, αIIbβ3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with αIIbβ3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant αIIbβ3 Ser753Pro, or β3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that αIIbβ3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of β3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of β3 Ser753Pro or αIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin β-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.

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Lee, Stella Y., Marc Mansour, and Bill Pohajdak. "B2-1, a Sec7- and Pleckstrin Homology Domain-Containing Protein, Localizes to the Golgi Complex." Experimental Cell Research 256, no. 2 (May 2000): 515–21. http://dx.doi.org/10.1006/excr.2000.4845.

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Sells, Mary Ann, Justin T. Barratt, Juliane Caviston, Sabine Ottilie, Ekkehard Leberer, and Jonathan Chernoff. "Characterization of Pak2p, a Pleckstrin Homology Domain-containing, p21-activated Protein Kinase from Fission Yeast." Journal of Biological Chemistry 273, no. 29 (July 17, 1998): 18490–98. http://dx.doi.org/10.1074/jbc.273.29.18490.

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31

Chen, Grace, Ioannis D. Dimitriou, Jose La Rose, Subburaj Ilangumaran, Wen-Chen Yeh, Gina Doody, Martin Turner, Jennifer Gommerman, and Robert Rottapel. "The 3BP2 Adapter Protein Is Required for Optimal B-Cell Activation and Thymus-Independent Type 2 Humoral Response." Molecular and Cellular Biology 27, no. 8 (February 5, 2007): 3109–22. http://dx.doi.org/10.1128/mcb.01014-06.

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ABSTRACT 3BP2 is a pleckstrin homology domain- and Src homology 2 (SH2) domain-containing adapter protein that is mutated in the rare human bone disorder cherubism and which has also been implicated in immunoreceptor signaling. However, a function for this protein has yet to be established. Here we show that mice lacking 3BP2 exhibited a perturbation in the peritoneal B1 and splenic marginal-zone B-cell compartments and diminished thymus-independent type 2 antigen response. 3BP2−/− B cells demonstrated a proliferation defect in response to antigen receptor cross-linking and a heightened sensitivity to B-cell receptor-induced death via a caspase-3-dependent apoptotic pathway. We show that 3BP2 binds via its SH2 domain to the CD19 signaling complex and is required for optimum Syk phosphorylation and calcium flux.

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32

RÜMENAPP, Ulrich, Andrea FREICHEL-BLOMQUIST, Burkhard WITTINGHOFER, Karl H. JAKOBS, and Thomas WIELAND. "A mammalian Rho-specific guanine-nucleotide exchange factor (p164-RhoGEF) without a pleckstrin homology domain." Biochemical Journal 366, no. 3 (September 15, 2002): 721–28. http://dx.doi.org/10.1042/bj20020654.

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Rho GTPases, which are activated by specific guanine-nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a recently cloned human cDNA, namely KIAA0337, encoding a protein containing 1510 amino acids (p164). It contains a RhoGEF-specific Dbl homology (DH) domain but lacks their typical pleckstrin homology domain. The expression of the mRNA encoding p164 was found to be at least 4-fold higher in the heart than in other tissues. Recombinant p164 interacted with and induced GDP/GTP exchange at RhoA but not at Rac1 or Cdc42. p164-ΔC and p164-ΔN are p164 mutants that are truncated at the C- and N-termini respectively but contain the DH domain. In contrast with the full-length p164, expression of p164-ΔC and p164-ΔN strongly induced actin stress fibre formation and activated serum response factor-mediated and Rho-dependent gene transcription. Interestingly, p164-ΔN2, a mutant containing the C-terminus but having a defective DH domain, bound to p164-ΔC and suppressed the p164-ΔC-induced gene transcription. Overexpression of the full-length p164 inhibited M3 muscarinic receptor-induced gene transcription, whereas co-expression with Gβ1γ2 dimers induced transcriptional activity. It is concluded that p164-RhoGEF is a Rho-specific GEF with novel structural and regulatory properties and predominant expression in the heart. Apparently, its N- and C-termini interact with each other, thereby inhibiting its GEF activity.

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Oak, Shilpa A., and Harry W. Jarrett. "Oligomerization of Mouse α1-Syntrophin and Self-Association of Its Pleckstrin Homology Domain 1 Containing Sequences†." Biochemistry 39, no. 30 (August 2000): 8870–77. http://dx.doi.org/10.1021/bi0000824.

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34

Kohn, A. D., K. S. Kovacina, and R. A. Roth. "Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase." EMBO Journal 14, no. 17 (September 1995): 4288–95. http://dx.doi.org/10.1002/j.1460-2075.1995.tb00103.x.

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Xu, Shunbin, Rahim Ladak, Deborah A. Swanson, Anna Soltyk, Hui Sun, Lynda Ploder, Danka Vidgen, et al. "PHR1 Encodes an Abundant, Pleckstrin Homology Domain-containing Integral Membrane Protein in the Photoreceptor Outer Segments." Journal of Biological Chemistry 274, no. 50 (December 10, 1999): 35676–85. http://dx.doi.org/10.1074/jbc.274.50.35676.

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Zhang, Lingqiang, Guichun Xing, Yi Tie, Ying Tang, Chunyan Tian, Li Li, Libo Sun, Handong Wei, Yunping Zhu, and Fuchu He. "Role for the pleckstrin homology domain-containing protein CKIP-1 in AP-1 regulation and apoptosis." EMBO Journal 24, no. 4 (February 10, 2005): 766–78. http://dx.doi.org/10.1038/sj.emboj.7600532.

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37

Klippel, A., W. M. Kavanaugh, D. Pot, and L. T. Williams. "A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain." Molecular and Cellular Biology 17, no. 1 (January 1997): 338–44. http://dx.doi.org/10.1128/mcb.17.1.338.

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Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.

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Yamamoto, Eiji, Jan Domański, Fiona B. Naughton, Robert B. Best, Antreas C. Kalli, Phillip J. Stansfeld, and Mark S. P. Sansom. "Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes." Science Advances 6, no. 8 (February 2020): eaay5736. http://dx.doi.org/10.1126/sciadv.aay5736.

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Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.

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Marshall, A. J., T. Zhang, and M. Al-Alwan. "Regulation of B-lymphocyte activation by the PH domain adaptor protein Bam32/DAPP1." Biochemical Society Transactions 35, no. 2 (March 20, 2007): 181–82. http://dx.doi.org/10.1042/bst0350181.

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PI3Ks (phosphoinositide 3-kinases) play critical roles in BCR (B-cell receptor) signalling via the generation of 3-phosphoinositide second messengers. Recruitment of PH domain (pleckstrin homology domain)-containing signal transduction proteins to the plasma membrane through binding to 3-phosphoinositide second messengers represents a major effector mechanism for PI3Ks. Here, we review data on the PH domain-containing adaptor protein Bam32 (B-cell adaptor molecule of 32 kDa)/DAPP1 (dual adaptor for phosphotyrosine and 3-phosphoinositides 1), focusing on its functions in B-lymphocyte activation. Present results support the view that Bam32/DAPP1 mediates multiple PI3K-dependent responses in B-cells through membrane-proximal mechanisms involving Src kinases, Rac1, F-actin and mitogen-activated protein kinases, resulting in selective effects on BCR-mediated proliferation, antigen presentation and generation of antibody responses.

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Bender, L., H. S. Lo, H. Lee, V. Kokojan, V. Peterson, and A. Bender. "Associations among PH and SH3 domain-containing proteins and Rho-type GTPases in Yeast." Journal of Cell Biology 133, no. 4 (May 15, 1996): 879–94. http://dx.doi.org/10.1083/jcb.133.4.879.

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The src homology region 3 (SH3) domain-bearing protein Bem1p and the Rho-type GTPase Cdc42p are important for bud emergence in Saccharomyces cervisiae. Here, we present evidence that through its second SH3 domain, Bem1p binds to the structurally and functionally similar proteins Boi1p and Boi2p, each of which contain an SH3 and pleckstrin homology (PH) domain. Deletion of BOI1 and BO12 together leads to impaired morphogenesis and poor ability. A PH domain-bearing segment of Boi1p that lacks the Bem1p-binding site is necessary and sufficient for function. This segment of Boi1p displays a two-hybrid interaction with Cdc42p, suggesting that Boi1p either binds directly to or is part of a larger complex that contains Cdc42p. Consistent with these possibilities, overexpression of Boi1p inhibits bud emergence, but this inhibition is counteracted by cooverexpression of Cdc42p. Increased expression of the Rho-type GTPase Rho3p, which is implicated in bud growth defects of boil boi2 mutants, suggesting that Boi1p and Boi2p may also play roles in the activation or function of Rho3p. These findings provide an example of a tight coupling in function between PH domain-bearing proteins and both Rho-type GTPases and SH3 domain-containing proteins, and they raise the possibility that Boi1p and Boi2 play a role in linking the actions of Cdc42p and Rho3p.

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41

Meller, Nahum, M. Jody Westbrook, John D. Shannon, Chittibabu Guda, and Martin A. Schwartz. "Function of the N-terminus of zizimin1: autoinhibition and membrane targeting." Biochemical Journal 409, no. 2 (December 21, 2007): 525–33. http://dx.doi.org/10.1042/bj20071263.

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Rho family small GTPases are critical regulators of multiple cellular functions. Dbl-homology-domain-containing proteins are the classical GEFs (guanine nucleotide exchange factors) responsible for activation of Rho proteins. Zizimin1 is a Cdc42-specific GEF that belongs to a second family of mammalian Rho-GEFs, CZH [CDM (Ced-5/DOCK180/Myoblast city)-zizimin homology] proteins, which possess a novel type of GEF domain. CZH proteins can be divided into a subfamily related to DOCK 180 and a subfamily related to zizimin1. The two groups share two conserved regions named the CZH1 (or DHR1) domain and the CZH2 (DHR2 or DOCKER) domains, the latter exhibiting GEF activity. We now show that limited proteolysis of zizimin1 suggests the existence of structural domains that do not correspond to those identified on the basis of homologies. We demonstrate that the N-terminal half binds to the GEF domain through three distinct areas, including the CZH1, to inhibit the interaction with Cdc42. The N-terminal PH (pleckstrin homology) domain binds phosphoinositides and mediates zizimin1 membrane targeting. These results define two novel functions for the N-terminal region of zizimin1.

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Dong, Lily Q., Hongyan Du, Sarah G. Porter, Lee F. Kolakowski, Adrian V. Lee, Lawrence J. Mandarino, Jianbing Fan, Douglas Yee, and Feng Liu. "Cloning, chromosome localization, expression, and characterization of an Src homology 2 and pleckstrin homology domain-containing insulin receptor binding protein hGrb10γ." Journal of Biological Chemistry 273, no. 7 (February 1998): 4288. http://dx.doi.org/10.1016/s0021-9258(17)47202-4.

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Dong, Lily Q., Hongyan Du, Sarah G. Porter, Lee F. Kolakowski, Adrian V. Lee, J. Mandarino, Jianbing Fan, Douglas Yee, and Feng Liu. "Cloning, Chromosome Localization, Expression, and Characterization of an Src Homology 2 and Pleckstrin Homology Domain-containing Insulin Receptor Binding Protein hGrb10γ." Journal of Biological Chemistry 272, no. 46 (November 14, 1997): 29104–12. http://dx.doi.org/10.1074/jbc.272.46.29104.

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Zhou, Qiong L., Zhen Y. Jiang, Allan S. Mabardy, Claudia M. Del Campo, David G. Lambright, John Holik, Kevin E. Fogarty, et al. "A Novel Pleckstrin Homology Domain-containing Protein Enhances Insulin-stimulated Akt Phosphorylation and GLUT4 Translocation in Adipocytes." Journal of Biological Chemistry 285, no. 36 (June 28, 2010): 27581–89. http://dx.doi.org/10.1074/jbc.m110.146886.

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Funamoto, Satoru, Kristina Milan, Ruedi Meili, and Richard A. Firtel. "Role of Phosphatidylinositol 3′ Kinase and a Downstream Pleckstrin Homology Domain–Containing Protein in Controlling Chemotaxis inDictyostelium." Journal of Cell Biology 153, no. 4 (May 14, 2001): 795–810. http://dx.doi.org/10.1083/jcb.153.4.795.

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We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3′ kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain–containing proteins. We demonstrate that in response to chemoattractants three PH domain–containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

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Safi, Alexias, Marie Vandromme, Sabine Caussanel, Laure Valdacci, Dominique Baas, Marc Vidal, Gilbert Brun, Laurent Schaeffer, and Evelyne Goillot. "Role for the Pleckstrin Homology Domain-Containing Protein CKIP-1 in Phosphatidylinositol 3-Kinase-Regulated Muscle Differentiation." Molecular and Cellular Biology 24, no. 3 (February 1, 2004): 1245–55. http://dx.doi.org/10.1128/mcb.24.3.1245-1255.2004.

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ABSTRACT In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.

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Imoto, M., I. Tachibana, and R. Urrutia. "Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p." Journal of Cell Science 111, no. 10 (May 15, 1998): 1341–49. http://dx.doi.org/10.1242/jcs.111.10.1341.

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Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

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Raabe, Thomas, Juan Riesgo–Escovar, Xiangdong Liu, Burkhard S. Bausenwein, Peter Deak, Peter Maröy, and Ernst Hafen. "DOS, a Novel Pleckstrin Homology Domain–Containing Protein Required for Signal Transduction between Sevenless and Ras1 in Drosophila." Cell 85, no. 6 (June 1996): 911–20. http://dx.doi.org/10.1016/s0092-8674(00)81274-x.

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Komander, David, Maria Deak, Nick Morrice, and Daan M. F. van Aalten. "Purification, crystallization and preliminary X-ray diffraction of a proteolytic fragment of PDK1 containing the pleckstrin homology domain." Acta Crystallographica Section D Biological Crystallography 60, no. 2 (January 24, 2004): 314–16. http://dx.doi.org/10.1107/s0907444903028518.

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Diggins, Nicole L., and Donna J. Webb. "APPL1 is a multifunctional endosomal signaling adaptor protein." Biochemical Society Transactions 45, no. 3 (June 15, 2017): 771–79. http://dx.doi.org/10.1042/bst20160191.

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Endosomal adaptor proteins are important regulators of signaling pathways underlying many biological processes. These adaptors can integrate signals from multiple pathways via localization to specific endosomal compartments, as well as through multiple protein–protein interactions. One such adaptor protein that has been implicated in regulating signaling pathways is the adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1). APPL1 localizes to a subset of Rab5-positive endosomes through its Bin–Amphiphysin–Rvs and PH domains, and it coordinates signaling pathways through its interaction with many signaling receptors and proteins through its PTB domain. This review discusses our current understanding of the role of APPL1 in signaling and trafficking, as well as highlights recent work into the function of APPL1 in cell migration and adhesion.

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Journal articles on the topic 'PLEKHA1 [Pleckstrin homology domain containing'

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