PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

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 actindependent spreading only after stimulation of the T-cell antigen receptor or the integrin ␣4␤1. 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 membraneassociated phosphatidylinositols generated by PI3K, whereby it coordinates with the actin cytoskeleton in lymphocyte spreading and immune synapse formation. IntroductionPleckstrin-1, the platelet and leukocyte C kinase substrate, is the major substrate of protein kinase C (PKC) in platelets, monocytes, macrophages, lymphocytes, and granulocytes. 1 Its 350 amino acid sequence can be divided into 3 motifs: PH domains at the aminoand carboxy-termini of the molecule and an intervening DEP domain. 2-4 A short stretch of amino acids between the aminoterminal PH domain and the DEP domain contains 3 sites of phosphorylation (Ser113, Thr114, and Ser117) by PKC, which are essential for the function of pleckstrin-1. [5][6][7] PH domains have been identified in approximately 252 other human proteins (Simple Modular Architecture Research Tool [SMART] database) and are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. 8 Previous results have suggested that PH domains mediate binding of their host proteins to certain phosphoinositides. [9][10][11] Consistent with this hypothesis, nearly all PH domain-containing proteins require membrane association for their function in signal transduction, including pathways that contribute to cytoskeletal assembly, membrane budding, and fusion. 4,7,[12][13][14][15] DEP homology domains are present in numerous signaling proteins, 3,16 including 67 found in the human genome (SMART database). The DEP domain is a protein module of approximately 100 amino acids, first found in the signaling proteins disheveled, Egl-10, and pleckstrin. 3 The domain is also present in certain kinases, regulators of G-protein signaling proteins (RGSs), and Epac, the cyclic adenosine monophosphateregulated guanine nucleotide ...

Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis

“…In T cells, plek2 functions in cytoskeleton reorganization, 23,31 whereas its roles in other hematopoietic lineages, especially erythroid cell, are completely unknown. During culture of mouse fetal liver erythroblasts, plek2 reached the highest protein levels on Day 1 in culture, and decreased on Day 2 (Figure 2A).…”

“…Since plek2 was reported to be membrane bound 23 with a putative function in actin rearrangement in other cell types, 31 we next tested the sub-cellular localization of plek2 in the cultured mouse fetal liver erythroblasts with confocal microscopy. Consistent with previous reports, plek2 localized primarily on the plasma membrane in the early stage of terminal erythropoiesis ( Figure 3A Figure 3A).…”

“…Among these genes, we identified pleckstrin-2 (plek2), which was previously reported to be involved in T-cell cytoskeleton reorganization, 23 play critical roles in terminal erythropoiesis. We demonstrated that plek2 is important in the regulation of actin cytoskeleton, cell differentiation and apoptosis.…”

“…Plek2 was reported to regulate actin cytoskeleton rearrangement 31,47 and T-cell cytoskeleton reorganization. 23 It contains two pleckstrin homology (PH) domains and one disheveled-Egl-10-pleckstrin (DEP) domain, which are present in a broad spectrum of proteins involved in cellular signaling and cytoskeleton organization. 48 These domains are important for their membrane association as evidenced in T cells, where plek2 is mostly localized on the plasma membrane.…”

“…48 These domains are important for their membrane association as evidenced in T cells, where plek2 is mostly localized on the plasma membrane. 23 However, the roles of plek2 in other lineages of hematopoietic cells, especially erythroid cells, are completely unknown. In this paper, we discovered a novel and critical role of plek2 in erythroid cells by regulating erythroid cell actin cytoskeleton dynamics and apoptosis through its interaction with cofilin.…”