Stimulation of phosphoinositide-hydrolysing phospholipase C (PLC) generating inositol-1,4,5-trisphosphate is a major calcium signalling pathway used by a wide variety of membrane receptors, activating distinct PLC-beta or PLC-gamma isoforms. Here we report a new PLC and calcium signalling pathway that is triggered by cyclic AMP (cAMP) and mediated by a small GTPase of the Rap family. Activation of the adenylyl cyclase-coupled beta2-adrenoceptor expressed in HEK-293 cells or the endogenous receptor for prostaglandin E1 in N1E-115 neuroblastoma cells induced calcium mobilization and PLC stimulation, seemingly caused by cAMP formation, but was independent of protein kinase A (PKA). We provide evidence that these receptor responses are mediated by a Rap GTPase, specifically Rap2B, activated by a guanine-nucleotide-exchange factor (Epac) regulated by cAMP, and involve the recently identified PLC-epsilon isoform.

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (I␣, I␤, and I␥) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP 2 synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP 2 synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA-and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP 2 by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP 2 synthesis has a central position in signaling by these three Rho GTPases.Synthesis and turnover of the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP 2 ),

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Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions.