The mechanism by which protein kinase A (PKA) inhibits G␣ q -stimulated phospholipase C activity of the  subclass (PLC) is unknown. We present evidence that phosphorylation of PLC 3 by PKA results in inhibition of G␣ q -stimulated PLC 3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLC 3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. Ligand stimulation of seven transmembrane domain receptors coupled to G␣ proteins of the G␣ q or G␣ i subfamilies results in the activation of the respective heterotrimeric G␣␥ protein complexes. Free G␣ q or G␥ subunits activate PLC 1 isoforms to catalyze the production of IP 3 and diacylglycerol from phosphatidylinositide 4,5-bisphosphate (1-3). PLC 1-4 comprise the currently known mammalian phosphatidylinositide-specific PLC subfamily. Although all PLCs are activated by G␣ q , PLC 2 and PLC 3 are also stimulated by G␥, primarily released from G␣ i (1).Cross-talk between the G protein-PLC pathway and PKA has been documented in numerous studies (4 -13). Although it is generally agreed that G protein-activated PLC activity can be inhibited by PKA (4 -11), PKA can enhance the G protein-PLC pathway in some cases (12, 13). Because PKA can inhibit phosphatidylinositide (PI) turnover activated by both G␣ q (4 -8) and G␣ i (9 -11) coupled receptors, it may inhibit the stimulation of both G␣ q -and G␥-stimulated PLC activity. This notion is further supported by studies with the G protein activators GTP␥S and AlF 4 Ϫ . These two compounds nonselectively activate all heterotrimeric G proteins and generate free G␣ and G␥ subunits that can stimulate PLCs. PKA inhibition of PI turnover initiated by GTP␥S or AlF 4 Ϫ (5, 8, 14, 15) is consistent with the inhibition of G␣q-as well as G␥-stimulated PLC activity. In addition, this phenomenon also suggests that the PKA effect is distal to receptors.Recently, the mechanism for PKA inhibition of G␥-stimulated PI turnover has been elucidated. Phosphorylation of PLC 2 by PKA resulted in inhibition of G␥-stimulated PI turnover (10). However, in the same study, PKA apparently did not inhibit G␣ 15 -and G␣ 16 -stimulated endogenous PLC ( 1 and  3 ) activity. More recently, Ali et al. (11) have reported phosphorylation of PLC 3 in response to CPT-cAMP treatment in RBL-2H3 cells expressing only PLC 3 . CPT-cAMP inhibited G␥-stimulated PLC 3 activated by the G␣ i -coupled formylmethionylleucylphenylalanine receptor but had no effect on PAFstimulated PLC 3 activity, presumably mediated by G␣ q . These studies led to the conclusions that phosphorylation of PLC 2 and PLC 3 by PKA could explain the inhibition of G␥-stimulated PI turnover by cAMP (10, 11). However, a biochemical mechanism for the inhibition by PKA of G␣ q -stimulated PLC activity observed in several systems remains to be clarified. In this study, we present evidence that phosphorylation of PLC 3 Ser 1105 by PKA results in d...
Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLC) pathway. In turn, phosphorylation of PLC by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca 2؉ release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both G␣ q -coupled (oxytocin and M1 muscarinic) and G␣ i -coupled (formylMet-Leu-Phe) receptor-stimulated PI turnover by 50 -100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLC 3 . Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formylMet-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLC 3 was stimulated directly by G␣ q or G␥ in overexpression assays. PKC phosphorylated PLC 3 at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLC 3 and site-directed mutagenesis identified Ser 1105 as the predominant phosphorylation site. Ser 1105 is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of G␣ q -stimulated PLC 3 activity was completely abolished by mutation of Ser 1105 to Ala. In contrast, mutation of Ser 1105 or Ser 26 , another putative phosphorylation target, to Ala had no effect on inhibition of G␥-stimulated PLC 3 activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit G␣ q -stimulated PLC 3 as a result of phosphorylation of Ser 1105 . Moreover, PKC and PKA both inhibit G␥-stimulated activity by mechanisms that do not involve Ser 1105 .Stimulation of seven transmembrane receptors coupled to the G␣ q or G␣ i subunits of heterotrimeric G proteins results in activation of PLC 1 isoforms that hydrolyze phosphatidylinositol 4,5-bisphosphate to generate the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (1, 2). IP 3 binds to a receptor in endoplasmic reticulum and releases intracellular calcium from its stores. Diacylglycerol, alone or in conjunction with elevated intracellular calcium, activates PKC and initiates additional cellular responses (3). Currently, four isoforms of mammalian PLC have been identified and characterized (4 -10). Significantly, PLC 3 is ubiquitously expressed and activated by all known PLC activators (G␣ q , G␥, and calcium) (2). Regulation of PLC 3 may be of great importance in many cellular processes (11-15). Insufficient expression of PLC 3 has been correlated with increased sensitivity to tumor formation (15, 16), whereas overexpression of PLC 3 seems to suppress tumor growth (17). PLC 3 knockout mice exhibit altered response to -opioids (11) or early embryonic lethality (18).Phosphorylation appears to play an important role in regulating the activity of PLC isoforms. Phosphorylation of PLC 3 or PLC 2 by PKA inhibits their activity and establishes a mechanism for cross-talk betwee...
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