Seven unrelated patients with atypical variants of hemizygous Fabry's disease were found among 230 men with left ventricular hypertrophy (3 percent). Fabry's disease should be considered as a cause of unexplained left ventricular hypertrophy.
Ras (Ha-Ras). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein.The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTP␥S⅐GST-HaRas. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTP␥S⅐GST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.Ras (Ha-Ras, Ki-Ras, N-Ras) is a signal-transducing guanine nucleotide-binding protein for tyrosine kinase-type receptors such as epidermal growth factor receptors and the Src family, leading to a mitogenic response and differentiation (for reviews, see Refs. 1 and 2). Ras has GDP-bound inactive and GTP-bound active forms, the latter of which makes physical contact with targets. Intensive investigations revealed that the Raf kinase family, consisting of c-Raf-1 (for reviews, see Refs. 3 and 4), A-Raf (5), and B-Raf (6 -9), is one of the direct targets for Ras. The activated Raf phosphorylates MAP 1 kinase kinase and activates it. Consequently the activated MAP kinase kinase activates MAP kinase, leading to the expression of certain genes such as c-fos (for reviews, see Refs. 10 and 11). Several molecules interacting with activated Ras in addition to Raf have been identified in mammals. These include phosphatidylinositol-3-OH kinase (12), Ral GDS (13,14), and Rin1 (15). On the basis of these observations, a variety of Ras targets may account for the pleiotropic functions of Ras. To understand the molecular mechanism of pleiotropic functions of Ras, it is essential to identify novel targets for Ras.In the present study, we discovered and partially purified another putative target for Ras with a molecular mass of about 180 kDa (p180) by use of GST-Ha-Ras affinity column chromatography and identified it as AF-6 (16), whose structure resembles that of Drosophila Canoe, which is involved in the Notch signaling pathway (17). S]methionine were purchased from DuPont NEN. A rabbit polyclonal antibody against a 16-mer peptide corresponding to 561-576 aa of human AF-6 (RVEQQPDYRRQESRTQ) was generated and purified. EXPERIMENTAL PROCEDURES Materials and Chemicals-AllPlasmid Construction-Plasmids, pGEX-Ha-Ras, pGEX-R-Ras, pGEX-RalA, and pGEX-RhoA were constructed as described previously (18). To obtain the in vitro translated N-terminal domain of AF-6 and Canoe, pRSET-AF-6 (36 -848 aa), pRSET-AF-6 (36 -206 aa), and pR-SET-Canoe (1-217 aa) were constructed as follows. The 2.4-kilobase cDNA fragment encoding AF-6 (36 -848 aa) was amplified by polymerase chain reaction from human brain Quick clone cDNA (Clontech Laboratories Inc., Palo Alto, CA). For the shorter N-terminal domain of ...
The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.
We examined the translocation of diacylglycerol kinase (DGK) ␣ and ␥ fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGK␣ and ␥ were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGK␥, but not DGK␣, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGK␥ was inhibited by the mutation of C1A but not C1B domain of DGK␥ and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGK␥ from the cytoplasm to the plasma membrane, whereas DGK␣ showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGK␥ and ␣ to the plasma membrane. The timing of the ATP-induced translocation of DGK␥ roughly coincided with that of PKC␥ re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKC␥ was obviously hastened by co-expression with DGK␥ and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction. Diacylglycerol (DG)1 is a second messenger regulating various cellular responses (1, 2). One of the important roles of DG is the activation of protein kinase C (PKC) (1, 3, 4). Thus, DG is very important for regulation of PKC activity and cellular response. DG is produced physiologically as a result of the signal-induced hydrolysis of phosphatidylinositol by phospholipase C and also from phosphatidylcholine by phospholipase D. Generated DG is phosphorylated to phosphatidic acid by diacylglycerol kinase (DGK) or cleavage by DG lipase (2, 5, 6). DGK is an important enzyme for inactivating PKC by attenuation of the DG level, contributing to regulation of the cellular response. In addition, phosphatidic acid itself activates PKC and PLC␥1 (7, 8) and modulates Ras GTPase-activating protein (9). DGKs have additional important functions for various cellular responses.To date at least nine subtypes of mammalian DGKs have been cloned and divided into five groups based on structure (2). Generally, all DGKs have cysteine-rich regions homologous to the C1A and C1B motifs of PKCs in the regulatory domain at the N terminus of the protein and possess a conserved catalytic domain in the C terminus of the protein. Type I DGKs, including DGK␣, -, and -␥, have EF-hand motifs and two cysteinerich regions in the regulatory domain (10 -12). Type II DGKs such as DGK␦ and -, have a pleckstrin homology domain instead of the EF-hand motif in addition to two cysteine-rich regions (13,14). Interestingly, the catalytic domains of DGK␦ and -are separated. Type III, consisting of DGK⑀, has only two cysteine-rich regions in the regulatory domain. Type IV, DGK and ...
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