Characterization of Rad, a new member of Ras/GTPase superfamily, and its regulation by a unique GTPase-activating protein (GAP)-like activity.
We have recently identified a new member of the Ras/GTPase superfamily termed Rad which has unique sequence features and is overexpressed in the skeletal muscle of humans with type II diabetes (Reynet, C., and Kahn, C. R. (1993) Science, 262, 1441-1444). When expressed in bacteria as a glutathione S-transferase fusion protein, Rad bound [alpha-32P]GTP quickly and saturably. Binding was specific for guanine nucleotides and displayed unique magnesium dependence such that both GTP and GDP binding were optimal at relatively high Mg2+ concentrations (1-10 mM). Rad had low intrinsic GTPase activity which was greatly enhanced by a GTPase-activating protein (GAP) activity present in various tissues and cell lines. Several known GAPs had no stimulatory effect toward Rad. Conversion of Ser to Asn at position 66 in Rad (equivalent to position 12 in Ras) resulted in a total loss of GTP binding. Mutation of Pro61 (equivalent to Gly12 in Ras) or Gln109 (equivalent to Gln61 in Ras) had no effect on Rad GTPase activity, whereas creation of a double mutation at these positions resulted in exceptionally high intrinsic GTPase activity. In vitro, Rad was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PK). Phosphopeptide mapping indicated two PKA phosphorylation sites near the COOH terminus. Rad also co-precipitated a serine/threonine kinase activity from extracts of various tissues and cell lines which catalyzed phosphorylation on Rad but was not inhibited by PKA inhibitor. Thus, Rad is a GTP-binding protein and a GTPase which has some structure/function similarities to Ras, but displays unique features. Rad may also be phosphorylated on serine/threonine residues by PKA and other kinases, as well as regulated by its own GAP which is present in many tissues and cell types.
Zhu
J
J
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215.
Reynet
C
C
Caldwell
J S
JS
Kahn
C R
CR
D000595:Amino Acid Sequence; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D011506:Proteins; C084355:RRAD protein, human; D020703:ras GTPase-Activating Proteins; D011494:Protein Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D018631:ras Proteins