The Role of the PIP5 Kinase Gamma 87 Isoform in the Regulation of the Actin Cytoskeleton

Phosphatidylinositol-4,5-bisphosphate (PIP2) is an important regulator of the actin cytoskeleton and plasma membrane functions. It is primarily synthesized by the type 1 phosphatidylinositol 4 phosphate 5 kinases (PIP5Ks). Mammals have three PIP5K genes (PIP5K alpha, PIP5K beta, and PIP5K gamma), and the gamma isoform has two ubiquitous 90 kDa and an 87 kDa splice variants. We found that the depletion of each PIP5K isoform individually by RNA interference (RNAi) or gene knockout by homologous recombination generated distinct changes in the actin cytoskeleton and signaling responses. The actin phenotype of the PIP5K gamma depletion (using pan siRNA, which is directed against a common sequence shared by the 90 and 87kDa isoforms) in HeLa cells is particularly striking: it results in increased actin stress fibers, decreased chemotaxis, and increased adhesion to fibronectin-coated substrates. There is also a striking increase in prominent focal adhesions (FA). Using real-time IRM, we found that the turnover of FA is 48% slower in the PIP5K gamma depleted cells. Likewise, there is a large decrease in the dynamic turnover of green fluorescent protein (GFP)-labeled vinculin and paxillin in FA, as monitored by fluorescence recovery after photobleaching. Since PIP5K gamma 90 has already been implicated in FA assembly, we depleted it specifically without depletion of the much more abundant PIP5K gamma 87 by using a PIP5K gamma 90 specific targeting sequence not found in PIP5K gamma 87. This fails to produce robust stress fibers. Overexpression of PIP5K gamma 87, but not the kinase dead enzyme, is able to rescue the pan PIP5K gamma knockdown actin phenotype in HeLa cells. Thus, PIP5K gamma 87 is the major contributor to the pan PIP5K gamma depletion/knockout robust actin and FA phenotype. Similar results were obtained in mouse embryonic fibroblasts (MEFs) from PIP5K gamma -/- mice. We sought to identify the molecular mechanisms of the PIP5K gamma depleted actin phenotype. Inhibitors of myosin, Rho-associated coiled-coil-containing protein kinase (ROCK), and RhoA GTPase all decreased the amount of thick actin stress fibers in PIP5K gamma RNAi cells, suggesting that the phenotype is due to abnormal RhoA activation. This is confirmed by the finding that RhoA activity is elevated in PIP5K gamma depleted/knock out cells. We hypothesize that PIP5K gamma regulates the actin cytoskeleton by inhibiting Rho, and thus its downstream effectors ROCK and myosin.