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.
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Research Article|
August 01 1998
Effects of phosphorylation on function of the Rad GTPase
Julie S. MOYERS;
Julie S. MOYERS
1Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, One Joslin Place, Boston, MA 02215, U.S.A.
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Jianhua ZHU;
Jianhua ZHU
1Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, One Joslin Place, Boston, MA 02215, U.S.A.
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C. Ronald KAHN
C. Ronald KAHN
1
1Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, One Joslin Place, Boston, MA 02215, U.S.A.
1To whom correspondence should be addressed (e-mail kahnr@joslab.harvard.edu).
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Publisher: Portland Press Ltd
Received:
December 05 1997
Revision Received:
April 20 1998
Accepted:
April 22 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 333 (3): 609–614.
Article history
Received:
December 05 1997
Revision Received:
April 20 1998
Accepted:
April 22 1998
Citation
Julie S. MOYERS, Jianhua ZHU, C. Ronald KAHN; Effects of phosphorylation on function of the Rad GTPase. Biochem J 1 August 1998; 333 (3): 609–614. doi: https://doi.org/10.1042/bj3330609
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