Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC⑀ in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC⑀ protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC⑀ activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras⅐c-Raf-1⅐PKC⑀ complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.The regulation of the Raf-1 serine/threonine kinase is a highly complex and poorly understood process (1, 2). Raf-1 activation is a critical component of the proliferative response. Raf-1 activation regulates the MAPK 1 cascade, a linear kinase cascade comprising the MAPKs, ERK1, and -2, which are the physiological targets for MEK1, the substrate for Raf-1 (3-5).Raf-1 can be activated in response to both oncogenes and receptor tyrosine kinase activation. Raf-1 activity is regulated by phosphorylation on serine and tyrosine residues (6 -8), with both PKC and Src implicated as Raf-1 regulatory kinases. Raf-1 activity is also subject to negative regulation by PKAmediated phosphorylation of serine 621 in the kinase domain (9, 10) and serine 43 in the Ras-binding domain (11), in response to elevations in intracellular cAMP levels. The direct interaction of Raf-1 with Ras (12, 13), plasma membrane phospholipids (14, 15), and molecular chaperone proteins of the hsp90 (16) and 14-3-3 families (17) Mason et al. (8) demonstrated that the expression of oncogenic Ras in cells induced phosphorylation of Raf-1 at serine 338, a consensus site for PKC-mediated phosphorylation, whereas activated Src induced Raf-1 phosphorylation predominantly at tyrosine 341; Raf-1 phosphorylation was also dependent upon its association with the plasma membrane. Mitogen-induced Raf-1 activity required phosphorylation on both Ser-338 and Tyr-341, which cooperate to induce full Raf-1 activation. Under specific conditions, Tyr-341 phosphorylation appeared to direct Ser-338 phosphorylation, since Raf-1 harboring a Y341A substitution was not phosphorylated on Ser-338 in cells expressing activated Ras and Src. Although coexpression of activated Ras and Src induced Ser...

C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serumfree medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) Oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serumindependent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor ␣ and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.The Ras GTPases are critical transducers of signals that are initiated from plasma membrane receptor tyrosine kinases and terminate in the nucleus as changes in gene expression. The c-Ras 1 proteins are implicated in the regulation of multiple biological processes as diverse as cell proliferation, migration, and differentiation. In most instances, the expression of mutated, oncogenic forms of Ras results in cell transformation. The loss of normal growth control may result in the acquisition of anchorage-independent growth and a diminished requirement for exogenous serum or growth factors (1) and are intimately related to the ability of Ras to stimulate the MAPK cascade, a critical component of the proliferative response (2-6). Deregulated cell proliferation is thought to be one consequence of activated Ras signaling directly and persistently through the Raf kinases, upstream mediators of the MAPK cascade. Ras transformation can also be accompanied by the synthesis and secretion of various growth factors, including interleukin 1␣, interleukin 6 (7), TGF-␣ (8 -10), vascular endothelial growth factor (VEGF) (11), and heparin-binding EGF (HB-EGF) (12). The production of autocrine growth factors may therefore be a potentially significant mechanism contributing to the ability of activated Ras proteins to undermine the processes that regulate normal cell proliferation.We recently reported that mouse fibroblasts transformed by the minimal expression of activated (G12V)Ha-Ras (V12H10 cells) were unable to proliferate in serum-free medium in the presence of suramin, a growth factor antagonist (13-15). ...

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf-1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pakmediated phosphorylation of S338 were examined by substitution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N-terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C-terminal glutamates and bulky C-terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is significant in epidermal growth factor-and Src-mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf-1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf-1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf-1 activation required for fulfillment of different biological roles within the cell. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Chitinase activity was detected in the supernatant fraction of a high-speed centrifugation preparation of broken Candida albicans yeast cells. The enzyme showed peak activity during the rapid budding phase of growth and was found to parallel the chitin synthase activity. The optimum conditions for the hydrolysis of chitin, regenerated from acetylation of chitosan, were determined. Analysis of the kinetics of the enzyme-substrate interaction and a measurement of their binding suggests that an equilibrium binding situation exists and that the kinetics follow a Langmuir isotherm interaction.