Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes ␤, ␥, and ␦, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLC⑀, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLC⑀ specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for HaRas is estimated to be approximately 40 nM, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLC⑀ induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLC⑀ is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLC⑀ is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLC⑀.

A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activaition. However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOHterminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa. cerevisiae cyclase rendered the catalytic domain of the Sc. pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein. Thus the leucine-rich repeats and the COOH terminus of the Sa. cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins.The ras oncogenes were identified as the transforming genes of retroviruses and the physiological function of ras proteins still remains to be elucidated (for review, see ref.

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A Schizosaccharomyces pombe gene encoding adenylate cyclase has been cloned by cross-hybridization with the Saccharomyces cerevisiae adenylate cyclase gene. (3)(4)(5). Although the precise mode of interaction between RAS proteins and yeast adenylate cyclase is unknown, regulatory proteins of adenylate cyclase appear to have somehow switched during the course of evolution. Specifically, it was shown that the cAMP concentration of the fission yeast Schizosaccharomyces pombe cells was not affected by the disruption or mutational activation of its sole ras gene, suggesting that its adenylate cyclase is not regulated by ras proteins (6). Sa. cerevisiae adenylate cyclase comprises 2026 amino acids in which the COOH-terminal 417 residues, called the catalytic domain, retains a Mn2+_ dependent cyclase activity (7). The remaining portion of the NH2 terminus was proposed to be essential for the RAS protein-and GTP-dependent activation in the presence of Mg2+ (7)(8)(9). In this report, we describe the primary structure of Sc. pombe adenylate cyclase.* Sequence comparison with Sa. cerevisiae adenylate cyclase reveals four segments displaying varying extents of homology. The functional significance of these segments is discussed. MATERIALS AND METHODSCell Strains, Growth Media, and Transformation. A Sa. cerevisiae strain, T50-3A (MATa, leu2, his3, trpl, ura3, cyrl-2) was described (7). A Sc. pombe strain 972h-s was obtained from D. Beach, Cold Spring Harbor Laboratory. Culture media for yeast cells and method of yeast transformation were as described (2-4, 10).DNA and RNA Preparation and Hybridization Analyses.

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.Ras proteins are small guanine nucleotide-binding proteins that serve as molecular switches in regulation of cellular proliferation and differentiation by cycling between the active GTP-bound and the inactive GDP-bound forms (for a review, see Ref. 1). In mammalian cells, the GTP-bound Ras exerts its action by physically associating with and activating effector proteins, such as the serine/threonine kinase Raf-1, through its effector region (amino acid residues 32-40 in human Ha-Ras). In addition to Raf-1 and its isoforms B-Raf and A-Raf, recent searches have identified a number of Ras effectors (or effector candidates) that associate directly with Ras in a GTP-dependent manner (for a review, see Ref.2). Two of them, RalGDS 1

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.Raf-1 is a serine/threonine kinase that plays a pivotal role in conveying a signal from receptor tyrosine kinases and Ras to the mitogen-activated protein kinase (MAPK) cascade. Although it is well established that Raf-1 interacts directly with the GTP-bound active form of Ras, the precise mechanism by which Raf-1 is activated by interaction with Ras is not known (for reviews, see references 1, 4, and 27). In addition to Raf-1, which is found in a variety of mammalian tissues, two close homologues, B-Raf and A-Raf, have been identified. These exhibit more limited tissue distribution: B-Raf expression is confined to the brain and testis, and A-Raf is expressed most abundantly in the ovary and epididymis (42). All Raf kinases possess three distinct regions designated conserved region 1 (CR1), CR2, and CR3 (for a review, see reference 10). The N-terminal regulatory domain contains CR1 and CR2, while the C-terminal CR3 corresponds to the protein kinase catalytic domain. CR1 consists of two distinct structural modules called the Ras-binding domain (RBD; residues 51 to 131) and the cysteine-rich domain (CRD; residues 139 to 184). RBD has a ubiquitin superfold (30) and constitutes a principal interface for GTP-dependent interaction with Ras and Rap1A (5, 46, 49). CRD consists of two zinc finger motifs resembling the C1 domain of protein kinase C, a phorbol ester-binding site (28). Constitutive activation of Raf-1 by N-terminal truncations indicated that the N-terminal reg...

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Phospholipase C is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase C is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase C. These results suggest a crucial role of phospholipase C downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phosphoinositide-specific phospholipase C (PLC) is a key event triggering intracellular signal transduction from various receptor molecules at the plasma membrane by yielding two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which induce activation of protein kinase C and mobilization of Ca 2ϩ from intracellular stores, respectively (9). Concurrently, reduction in PIP 2 concentration appears to be an important signal because activities of various actin-binding proteins and pleckstrin homology domain-containing proteins are modulated through interaction with PIP 2 (25). More than 12 mammalian PLC isoforms have been identified and organized into five classes (␤, ␥, ␦, ε, and ), which are regulated through distinct mechanisms (9). PLCε is characterized by possession of two Ras-associating domains and a CDC25 homology domain. The Ras-associating domains are responsible for activation of PLCε through direct association with the GTP-bound active forms of the small GTPases Ras (15, 23), Rap1 (23,24), and Rap2 (20). Stimulation of cells by growth factors, such as platelet-derived growth factor, induces persistent activation of PLCε through activation of Ras and Rap1 (24). The rapid and initial phase of this activation is mediated by Ras at the plasma membrane, whereas Rap1 is responsible for the prolonged activation mainly at the Golgi complex (24). The CDC25 homology domain acts as a guanine nucleotide exchange factor for Rap1 and is crucial for the prolonged activation of PLCε by Rap1 (14,24). The involvement of other factors, such as the ␣ subunits of the G 12 and G 13 families or the ␤ 1 ␥ 2 subunits ...