Cloning of bovine GAP and its interaction with oncogenic ras p21

Abstract

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.

Increasing complexity of Ras signaling

“…However, NMR structural analysis of these Ras variants suggest that the mutations produce structural changes remote from the site of mutation and thus these residues may not be directly involved in contacts with Raf-1 (Sharon Campbell, unpublished observations). The Q61L oncogenic mutation increases a nity for both GAPs (Vogel et al, 1988), consistent with the recently determined structure of Ras complexed with the catalytic domain of p120 Ras-GAP. Interestingly, whereas only WT Ras-GTP, binds to Raf-1 with high a nity leading to Raf-1 activation, both Q61L Ras-GDP and Q61L Ras-GTP bind Raf-1 with reasonable a nity and can lead to its activation (Moodie et al, 1995).…”

Identification of a Phosphatidylinositol 4,5-Bisphosphate-binding Site in Chicken Skeletal Muscle α-Actinin

“…The C2 domains of PKC (5, 6), phospholipase A 2 (28), PLC, Ras-GTPase activating protein (29), rabphilin (30), and synaptotagmin I (31, 32) have been proposed to contain phospholipid binding domains. ADP-ribosylation factor I (8), dynamin (33), myristoylated alanine-rich protein kinase C substrate (34), -calpain (7), and many actinregulating proteins (1, 10 -14) have also been shown to interact with acidic phospholipids including PIP 2 .…”

“…There has been another report that inositol 1,4,5-trisphosphate binds to PLC-␦1 and that this interaction is inhibited by PIP 2 (38). The inositol 1,4,5-trisphosphate binding site on PLC-␦1 is thought to comprise amino acids 30 -43, which overlaps with the site which we aligned (amino acids [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. In fact, the peptide from PLC-␦1 strongly inhibited the activity of PLC-␥1, but PEP-Grb7 did not inhibit.…”

Synaptotagmin V: a novel synaptotagmin isoform expressed in rat brain

“…It consists of a short amino-terminal intravesicular domain, a single transmembrane region and a larger cytoplasmic region [4]. The latter contains two internal repeats that are similar to the C2 domains found in several isoforms of protein kinase C, some phospholipases, rabphilin 3A, GTPase activating protein, brain protein Doc2 and the C. elegans protein unc-13 [8][9][10][11][12][13][14]. C2 domains confer Ca 2+-dependent phospholipid binding to at least some of these proteins.…”