Phosphorylation Of PIP2 Research Articles

Phosphorylation of PIP2;7 by CPK28 or Phytophthora kinase effectors dampens pattern-triggered immunity in Arabidopsis

Plasma membrane intrinsic proteins (PIPs), a subclass of aquaporins, play an important role in plant immunity by acting as H2O2 transporters. Their homeostasis is mostly maintained by C-terminal serine phosphorylation. However, the kinases that phosphorylate PIPs and manipulate their turnover are largely unknown. Here, we found that Arabidopsis thaliana PIP2;7 positively regulates plant immunity by transporting H2O2. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) directly interacts with and phosphorylates PIP2;7 at Ser273/276 to induce its degradation. During pathogen infection, CPK28 dissociates from PIP2;7 and destabilizes, leading to PIP2;7 accumulation. As a countermeasure, oomycete pathogens produce conserved kinase effectors that stably bind to and mediate the phosphorylation of PIP2;7 to induce its degradation. Our study identifies PIP2;7 as a novel substrate of CPK28 and shows that its protein stability is negatively regulated by CPK28. Such phosphorylation could be mimicked by Phytophthora kinase effectors to promote infection. Accordingly, we developed a strategy to combat oomycete infection using a phosphorylation-resistant PIP2;7S273/276A mutant. The strategy only allows accumulation of PIP2;7S273/276A during infection to limit potential side effects on normal plant growth.

Mice Lacking Pleckstrin or Pleckstrin-2 Each Have Unique Platelet Secretion Defects.

Approximately 1% of total cellular protein within platelets and leukocytes is pleckstrin, a protein best known for containing the two prototypic Pleckstrin Homology (PH) domains. Following platelet activation, PKC rapidly phosphorylates pleckstrin, inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Platelets also contain a widely expressed paralog of pleckstrin, called pleckstrin-2. Although the activity of pleckstrin is regulated through protein phosphorylation, pleckstrin-2 is not a phosphoprotein, but is instead activated by binding a specific PI3K generated phospholipid, phosphatidylinositol 3,4 bisphosphate (PI3,4P2). To define the role of individual pleckstrin isoforms in vivo, we generated mice containing loss of function mutations within either the pleckstrin or pleckstrin-2 genes. Mice lacking either isoform exhibited no spontaneous hemorrhage. Studies of pleckstrin knockout platelets demonstrated that they displayed normal aggregation and secretion in response to collagen and thrombin. In contrast, pleckstrin null platelets had no secretion of dense or alpha granules following exposure to the PKC stimulant, PMA. This demonstrates that pleckstrin is the critical effector for PKC-mediated platelet secretion. Our findings also suggest that an alternative pathway in pleckstrin knockout platelets is able to compensate for the secretion defect when the cells are stimulated with an agonist such as thrombin. We reasoned that the compensatory pathway might utilize another PIP2-dependent second messenger. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet secretion, we tested whether a PI3K-dependent pathway compensated for the loss of pleckstrin. We found that pleckstrin knockout platelets completely failed to secrete in response to stimulation of the thrombin receptor in the presence of a PI3K inhibitors (LY294002 or wortmannin.) This demonstrates that second messengers generated by PI3K are able to compensate for the secretion defect induced by the loss of pleckstrin. In contrast to the findings observed with pleckstrin knockout platelets, we have found that platelets lacking pleckstrin-2 aggregated and secreted normally in response to thrombin, collagen, and PMA. Additionally, unlike the effect seen on pleckstrin knockout platelets, inhibitors of PI3K had no effect on the aggregation or secretion of pleckstrin-2 knockout platelets. However, again in contrast to pleckstrin knockout platelets, pleckstrin-2 null platelets failed to secrete in response to thrombin when they were exposed to inhibitors of either PLC or PKC. This demonstrates that pleckstrin-2 knockout platelets compensate for their secretion defect by a pathway dependent on PLC and PKC. It is notable that PI3K or PKC inhibitors only minimally affected the thrombin-induced secretion of wild-type platelets unless both inhibitors were used together. Together, our results show that both pleckstrin and pleckstrin-2 are essential components of thrombin-mediated platelet secretion. These data also demonstrate that thrombin-induced platelet secretion can be mediated by one of two parallel pathways, the first involving PKC and pleckstrin, and the second involving PI3K and pleckstrin-2.

Knockout of the PKC Substrate Pleckstrin Causes Pleomorphic Defects in Platelets, Lymphocytes and Granulocytes.

Pleckstrin is a 40 kDa phosphoprotein containing amino- and carboxyl-terminal Pleckstrin Homology (PH) domains separated by a DEP domain. Pleckstrin's expression is restricted to platelets and leukocytes, and represents approximately 1% of total cellular protein within these cells. Following platelet and leukocyte activation, PKC rapidly phosphorylates pleckstrin inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Heterologously expressed phosphorylated pleckstrin colocalized with integrins and induces cytoskeletal reorganization. To better define the role of pleckstrin in vivo, we introduced a loss-of-function mutation into the murine pleckstrin gene. Pleckstrin-null mice were present in offspring at a frequency consistent with a Mendelian inheritance pattern. Adult pleckstrin −/− mice had 32% lower platelet counts than their littermates, but exhibited no spontaneous hemorrhage. Given the role of PKC and phospholipid second messengers on cytoskeletal dynamics, and our observations of pleckstrin overexpression in cell lines, we analyzed whether loss of pleckstrin affected cell spreading. Pleckstrin −/− platelets spread extremely poorly upon immobilized fibrinogen, and rarely exhibited broad membrane extensions. Granulocytes from pleckstrin −/− mice also have a spreading defect, as well as impaired ability to generate reactive oxygen species in the response to TNFα. Knockout B-cells, CD4-T-cells, and CD8-T-cells all migrated approximately 30% as efficiently as wild type cells in response to a gradient of SDF-1α in a transwell assay. These data suggest that loss of pleckstrin causes cytoskeletal defects in cells of multiple hematopoietic lineages. Analyzing whether this caused a functional defect, we found that pleckstrin −/− platelets exhibited a 22% dense- and 24% alpha-granule exocytosis defect, and a 35% defect in thrombin-induced calcium entry. In spite of these abnormalities, platelets changed shape and aggregated normally after stimulation with thrombin, ADP, or collagen in vitro. Pleckstrin knockout platelets did have a markedly impaired aggregation response following exposure to the PKC stimulant, PMA. This suggested that pleckstrin is a critical effector for PKC-mediated aggregation, but another pathway is able to compensate for this loss of pleckstrin following agonist stimulation. We reasoned that the alternative pathway might also utilize PIP2-dependent second messengers. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet aggregation, we tested whether PI3K compensated for the loss of pleckstrin. We found that the PI3K inhibitor, LY294002 profoundly impaired the aggregation of pleckstrin knockout platelets in response to stimulation of the thrombin receptor. In contrast, the PI3K inhibitor minimally affected wild type platelets. This demonstrates that second messengers generated by PI3K are able to compensate for loss of pleckstrin. This also demonstrates that thrombin-induced platelet aggregation can be mediated by one of two parallel pathways, one involving PKC and pleckstrin, and the other involving PI3K. Together, our results show that pleckstrin is an essential component of PKC-mediated platelet activation and signals directed to the cytoskeleton.

The relative role of PLCβ and PI3Kγ in platelet activation

Stimulation of platelet G protein-coupled receptors results in the cleavage of phosphatidylinositol 4,5-trisphosphate (PIP(2)) into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol by phospholipase C (PLCbeta). It also results in the phosphorylation of PIP2 by the gamma isoform of phosphatidylinositol 3-kinase (PI3Kgamma) to synthesize phosphatidylinositol 3,4,5-trisphosphate. To understand the role of PIP2 in platelet signaling, we evaluated knock-out mice lacking 2 isoforms of PLCbeta (PLCbeta2 and PLCbeta3) or lacking the G(betagamma)-activated isoform of PI3K (PI3Kgamma). Both knock-out mice were unable to form stable thrombi in a carotid injury model. To provide a functional explanation, knock-out platelets were studied ex vivo. PLCbeta2/beta3-/- platelets failed to assemble filamentous actin, had defects in both secretion and mobilization of intracellular calcium, and were unable to form stable aggregates following low doses of agonists. Platelets lacking PI3Kgamma disaggregated following low-dose adenosine diphosphate (ADP) and had a mildly impaired ability to mobilize intracellular calcium. Yet, they exhibited essentially normal actin assembly and secretion. Remarkably, both PLCbeta2/beta3-/- and PI3Kgamma-/- platelets spread more slowly upon fibrinogen. These results suggest substantial redundancy in platelet signaling pathways. Nonetheless, the diminished ability of knock-out platelets to normally spread after adhesion and to form stable thrombi in vivo suggests that both PLCbeta2/beta3 and PI3Kgamma play vital roles in platelet cytoskeletal dynamics.