Ken-ichi Kariya

Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMCs), sodium nitroprusside (SNP) (10(-7) to 10(-4) M), atrial natriuretic peptide (ANP) (10(-9) to 10(-6) M) and 8-bromo-cyclic GMP (10(-6) to 10(-3) M) inhibited the whole blood serum (WBS)-induced DNA synthesis by about 30%. The doses of SNP and ANP necessary for the inhibition of the WBS-induced DNA synthesis were similar to those necessary for the formation of cellular cyclic GMP (cGMP). These agents were effective even when added 6 h after stimulation of the cells with WBS. These results suggest that cGMP inhibits the proliferation of rabbit aortic SMCs by inhibiting the progression from the G1 into S phase of the cell cycle and raise the possibility that cGMP-elevating vasodilators may suppress the atherogenic process by inhibiting vascular SMC proliferation.

IDENTIFICATION OF PLC210, A CAENORHABDITIS ELEGANS PHOSPHOLIPASE C, AS A PUTATIVE EFFECTOR OF RAS

Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematodeCaenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras.

Possible involvement of protein kinase C in platelet-derived growth factor-stimulated DNA synthesis in vascular smooth muscle cells☆

In cultured rabbit aortic vascular smooth muscle cells (VSMC), protein kinase C-activating phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) stimulated DNA synthesis in the presence of 10% cell-free plasma-derived serum. This stimulation was half that shown by PDGF. 4 alpha-Phorbol-12,13-didecanoate, known to be inactive for protein kinase C, was without effect in stimulating DNA synthesis. Prolonged treatment of the cells with PDBu led to a marked decrease in protein kinase C. In the pDBu-treated cells, the TPA-stimulated DNA synthesis was completely abolished whereas the PDGF-stimulated DNA synthesis was decreased to about half that in the control cells. These results suggest that protein kinase C is involved in PDGF-stimulated proliferation of VSMC.

THE STRENGTH OF INTERACTION AT THE RAF CYSTEINE-RICH DOMAIN IS A CRITICAL DETERMINANT OF RESPONSE OF RAF TO RAS FAMILY SMALL GTPASES

ABSTRACT To be fully activated at the plasma membrane, Raf-1 must establish two distinct modes of interactions with Ras, one through its Ras-binding domain and the other through its cysteine-rich domain (CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and even antagonizes Ras-dependent activation of Raf-1. We proposed previously that this property of Rap1A may be attributable to its greatly enhanced interaction with Raf-1 CRD compared to Ras. On the other hand, B-Raf, another Raf family member, is activatable by both Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Raf CRD did not exhibit the enhanced interaction with Rap1A, suggesting that the strength of interaction at CRDs may account for the differential action of Rap1A on Raf-1 and B-Raf. The importance of the interaction at the CRD is further supported by a domain-shuffling experiment between Raf-1 and B-Raf, which clearly indicated that the nature of CRD determines the specificity of response to Rap1A: Raf-1, whose CRD is replaced by B-Raf CRD, became activatable by Rap1A, whereas B-Raf, whose CRD is replaced by Raf-1 CRD, lost its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is selectively enhanced was isolated and found to possess the double mutation K252E/M278T. B-Raf carrying this mutation was not activated by Rap1A but retained its response to Ras. These results indicate that the strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family small GTPases.

Differential Structural Requirements for Interaction of Ras Protein with Its Distinct Downstream Effectors

Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2 phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.

Effect of Phosphorylation on Activities of Rap1A to Interact with Raf-1 and to Suppress Ras-dependent Raf-1 Activation

Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152–184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.

Antiproliferative action of protein kinase C in cultured rabbit aortic smooth muscle cells.

In rabbit aortic smooth muscle cells (SMC), protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the whole blood serum (WBS)-induced DNA synthesis. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate (PDBu), but not by 4 alpha-phorbol-12,13- didecanoate known to be inactive for this enzyme. Prolonged treatment of the cells with PDBu caused the down-regulation of protein kinase C. In these cells, WBS still induced DNA synthesis but the inhibitory action of TPA was abolished. DNA synthesis started at 18 h and reached a maximal level 24 h after the addition of WBS. TPA inhibited the WBS-induced DNA synthesis even when added 12 h after the addition of WBS. These results suggest that protein kinase C has an antiproliferative action in rabbit aortic SMC and that this action is attributed to the inhibition of the progression from the late G1 into S phase of the cell cycle. TPA also inhibited the phospholipase C-mediated hydrolysis of phosphoinositides which was induced by WBS within several minutes, but the relevance of this effect on the antiproliferative action of TPA is uncertain.

Effect of Association with Adenylyl Cyclase-Associated Protein on the Interaction of Yeast Adenylyl Cyclase with Ras Protein

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells

Incubation of the [3H] inositol-labeled cultured rabbit vascular smooth muscle cells (VSMCs) with either endothelin or angiotensin II caused a rapid formation of inositol mono-, bis- and trisphosphates (IP1, IP2 and IP3, respectively). Time courses of the endothelin- and angiotensin II-induced formation of these inositol phosphates were similar. The maximal levels of IP1, IP2 and IP3 formation induced by endothelin were about 50%, 25% and 40%, respectively, of those induced by angiotensin II. The doses of endothelin necessary for the half maximal and maximal extents of the formation of IP1 were about 1 nM and 100 nM, respectively. Protein kinase C-activating 12-Q-tetradecanoylphorbol-13-acetate (TPA) inhibited the endothelin-induced formation of IP1 with the half maximal extent of inhibition seen at 3 nM. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate, but not by 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme. These results indicate that endothelin causes the phospholipase C-mediated hydrolysis of phosphoinositides, though to a lesser extent than angiotensin II, in cultured VSMCs and suggest that protein kinase C modulates the signaling mechanism of endothelin to the phospholipase C.

Coiled-coil Interaction of N-terminal 36 Residues of Cyclase-associated Protein with Adenylyl Cyclase Is Sufficient for Its Function in Saccharomyces cerevisiae Ras Pathway

In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to conferRAS2 Val-19-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif αXXαXXX (where α represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.