Effects of phosphorylation on function of the Rad GTPase

Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):609-14. doi: 10.1042/bj3330609.

Abstract

Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Calmodulin / metabolism
  • Casein Kinase II
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • GTP-Binding Proteins / metabolism
  • GTP-Binding Proteins / physiology*
  • Guanosine Triphosphate / metabolism
  • Humans
  • Hydrolysis
  • Kinetics
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Phosphotransferases (Alcohol Group Acceptor) / physiology*
  • Protein Kinase C / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • ras Proteins*

Substances

  • Calmodulin
  • RRAD protein, human
  • Guanosine Triphosphate
  • Phosphotransferases (Alcohol Group Acceptor)
  • guanosine phosphotransferase
  • Casein Kinase II
  • Protein-Serine-Threonine Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases
  • GTP-Binding Proteins
  • ras Proteins