Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.

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Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric G; proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat la cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic ml receptor (mlR) Raf-1 is a serine/threonine protein kinase which is an immediate effector of Ras GTP (1,35,36,40). Raf-1 regulates the mitogen-activated protein kinase (MAPK) pathway by phosphorylating and activating MEK (22), the dual threonine/ tyrosine recognition kinase which phosphorylates and activates MAPK (11). Raf-1 therefore plays a pivotal role in Ras GTPdependent conversion of growth factor receptor-stimulated tyrosine kinase activity to stimulation of cytoplasmic serine/ threonine protein kinases (22). Stimulation of Raf-1 activity is required for growth or differentiation of many different cell types. Control of Raf-1 activity will dramatically influence the responsiveness of cells to growth factors. Recently it has been shown that cyclic AMP (cAMP) activation of protein kinase A (PKA) inhibits growth factor stimulation of 10,15,28,39). The negative regulation of Raf-1 was independent of Ras * GTP loading. The implication is that the growth inhibitory effects of cAMP observed in many cell types are related to the PKA-catalyzed inhibition of Raf activation. The mechanism for the uncoupling of Raf activation from loading of Ras * GTP is unclear but is receiving attention from many laboratories.Current studies have demonstrated that forskolin stimulation of adenylyl cyclase or cAMP analogs inhibits growth factor stimulation of Raf. The suggestion is that hormonal regulation of cAMP synthesis could regulate Raf activation in response to growth factor receptor stimulation. Hormonal regulation of Raf activation in response to growth factors would indicate that the cAMP-mediated control of this pathway occurred

Gq is the heterotrimeric guanine nucleotidebinding protein that activates the 1B isoforms of phosphatidylinositol-specific phospholipase C (PI-PLC). The Gq a-subunit polypeptide (aq) was N-terminally modified by addition ofa 9-aa sequence, YPYDVPDYA. Placement of the 9-aa epitope tag at the N terminus allowed expression of functional aq polypeptides and selective identification of plasmid-expressed wild-type and mutant G-protein a subunits. Mutation of glutamine-209 to leucine in the N-terminally epitope-tagged aq (NePIaqQ209L) inhibited GTPase activity and persistently activated PI-PLC, resulting in high steady-state levels of inositol phosphates. The elevated levels of inositol phosphates resulting from NePIaqQ209L expression were similar to those obtained with carbachol activation of the M1 muscarinic acetylcholine receptor. The Gq-coupled M1 receptor, which stimulates PI-PLC activity, and phorbol esters, acting via protein kinase C, activate the cytoplasmic mitogen-activated protein kinase in COS cells. However, the constitutive activation of PI-PLC enzymatic activity resulting from expression of GTPase-deficient aq was unable to persistently activate this kinase. The results indicate that persistent PI-PLC activation is insufficient to sustain the stimulation of a cytoplasmic serine/threonine protein kinase regulated by Gq-coupled receptor signal-transduction pathways.Several phosphatidylinositol-specific phospholipase C (Pl-PLC) enzymes have been characterized. Stimulation of PT-PLC enzymes in response to a number ofligands and receptors results in the generation ofintracellular inositol trisphosphates and diacylglycerol (1,2). The M1 muscarinic acetylcholine receptor (M1R) is an example of a well-characterized receptor that robustly stimulates PI-PLC activity (3). The M1R stimulation of PI-PLC activity is transduced by the heterotrimeric guanine nucleotide-binding protein Gq (4). The a subunit of Gq (aq) and related members of the Gq family of a-subunit polypeptides stimulate the , isoforms of PI-PLC (4-6). This contrasts with the tyrosine-kinase growth factor receptors, which regulate the y isoform of 8).The ectopic expression and stimulation of the M1R and other receptors that couple to Gq and activate PI-PLC activity have been shown to induce mitogenic and tumorigenic responses in specific cell types (9-11). These results suggest that constitutively activated Gq might drive the signal-transduction pathways regulated by the M1R and stimulate mitogenic and/or tumorigenic responses, bypassing the requirement for persistent receptor stimulation. This prediction is based on the ability of constitutively activated mutants of the a, and ai polypeptides to persistently stimulate G.-and

Serpentine receptors coupled to the heterotrimeric G protein, Gi2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the Gi2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the Gi2-coupled thrombin and acetylcholine muscarinic M2 receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. Gi2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of a12 inhibits both the Raf-dependent and-independent pathways activated by G12-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Stel 1 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Stel 1 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by G12-coupled receptors as well as other receptor systems, including the tyrosine kinases.