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.

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Mitogen-activated protein (MAP) kinase is a widely expressed protein serine/threonine kinase that serves as a convergence point for many signaling pathways including receptor tyrosine kinases, G protein-coupled receptors, and protein kinase C (PKC). The hormonal regulation of MAP kinase was studied in cultured established rat inner medullary collecting tubule (RIMCT) cells. Neither vasopressin nor beta-adrenergic agonists stimulated MAP kinase, despite clear stimulation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. In contrast, carbachol, ATP, and epidermal growth factor (EGF), which are known to antagonize vasopressin action in the RIMCT, stimulated the MAP kinase pathway. This stimulation was mimicked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, which directly activates PKC. The potency with which EGF and carbachol activated MAP kinase was similar to the potency with which they inhibited vasopressin-stimulated cAMP accumulation. To assess the role of Gi proteins in these stimulatory events, RIMCT cells were pretreated with pertussis toxin to inhibit Gi-mediated signaling. Pertussis toxin did not influence ATP- or EGF-stimulated MAP kinase, but completely inhibited carbachol stimulation, suggesting that Gi proteins mediate muscarinic stimulation. Prolonged exposure of RIMCT cells to high phorbol ester concentrations to downregulate PKC ablated carbachol- and ATP-stimulated MAP kinase, but not EGF-stimulated MAP kinase, suggesting that PKC is a component of the network involved in MAP kinase activation by purinergic and muscarinic agonists. Investigation of the sidedness of the hormonal stimulations indicated that EGF-stimulated MAP kinase was highly polarized, occurring exclusively from the basolateral surface, whereas carbachol stimulated MAP kinase similarly from either cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

GTPase-inhibiting mutations of the a subunit (a12) of the G protein, Gj2, result in constitutive activation of a12 signal transduction functions. GTPase-inhibited a j2 mutant polypeptides, referred to as gip2 oncoproteins, have glutamine-205 mutated to leucine (a12Q205L). Expression of the a12Q205L polypeptide inhibits adenylyl cyclase stimulation, constitutively activates p42 mitogen-activated protein kinase, and transforms Rat la fibroblasts. The aI2 polypeptides are N-terminal-myristoylated, but the function of myristoylation is unclear in ae2 signal transduction. We have tested the requirement for myristoylation on the ability of the at2Q205L mutant polypeptide to constitutively regulate signal pathways and cell transformation. When expressed in Rat la cells, the nonmyristoylated ai2Q205L polypeptide is membrane associated but is unable to regulate adenylyl cyclase or p42 mitogen-activated protein kinase and does not induce cellular transformation. We conclude that myristoylation is absolutely necessary for ai2Q205L signal transduction and regulation of effector enzymes in the cell.Protein N-myristoylation involves the covalent attachment of a 14-carbon myristoyl fatty acid to the N-terminal glycine of a limited number of cellular proteins (1, 2). Among this group of proteins, cotranslational N-myristoylation appears to be important in protein localization and biological activity (2). Significant attention has been recently focused on the role of protein N-myristoylation because of its requirement for the fibroblast transforming ability of cellular oncoproteins such as v-Src and v-Abl (3-5). A subset of G protein a subunit family members, namely ail, aa2, ai3, a0, and a,, are N-terminal-myristoylated (6-8).It has been proposed that N-terminal amino acid residues and myristoylation of specific a subunits is involved in their membrane association (7)(8)(9)(10) would be similar to the proposed role of N-terminal myristoylation of v-Src and its interaction with cellular proteins required for Src signal transduction and cellular transformation by v-Src (3-6, 16). Until recently, it was difficult to approach the question of whether myristoylation was important in G protein a subunitmediated signal transduction. It has now been shown that GTPase-inhibiting mutations constitutively activate G protein a subunit-mediated signal transduction functions (17,18). One such mutation changes the glutamine-205 codon to a leucine codon in the ai2 gene (ai2Q205L). The GTPaseinhibited ai2Q205L mutant polypeptide has been found to constitutively inhibit adenylyl cyclase stimulation in several cell types (19,20). In addition, ai2Q205L is a tissue-selective oncogene referred to as gip2 (21), capable of causing transformation of Rat la fibroblasts (22, 23). The activated signal transducing ability of the gip2 oncoprotein also results in the constitutive stimulation of the p42 mitogen-activated protein kinase [p42 MAP kinase (24)]. Thus, the activated nature of the gip2 oncogene product provides the necessary dominant respons...