EFFECTS OF PHOSPHORYLATION ON FUNCTION OF THE RAD GTPASE

Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension t hat 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 278-247 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad i s complexed with calmodulin-dependent protein kinase II (CaM-KII) in C 2C12 cells. Here we show that Rad serves as a substrate for phosphoryl ation 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 anal ysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate mu ltiple C-terminal serine residues, including Ser(214), Ser(257), Ser(2 73), Ser(290) and Ser(299). Incubation of Rad with PKA decreases GTP b inding by 60-70 %, but this effect seems to be independent of phosphor ylation, as it is observed with the Ser(273) --> Ala mutant of Rad con taining a mutation at the site of PKA phosphorylation. The remainder o f the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metasta sis suppressor nm23. However, phosphorylation of Rad by PKC and CKII a bolishes the interaction of Rad with calmodulin. These findings sugges t that the binding of Rad to calmodulin, as well as its ability to bin d GTP, might be regulated by the activation of several serine kinases.