K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function.
Fine tuning of Ras activity is widely known as a mechanism to induce different cellular responses. Recently, we have shown that calmodulin (CaM) binds to K-Ras and that K-Ras phosphorylation inhibits its interaction with CaM. In this study we report that CaM inhibits K-Ras phosphorylation at Ser181 by protein kinase C (PKC) in vivo, and this is a mechanism to modulate K-Ras activity and signaling. Although CaM inhibition increased the activation of endogenous K-Ras, PKC inhibition decreased its activation status. We demonstrate that K-Ras phosphorylation decreased susceptibility to p120GAP activity. Accordingly, we also observed that non-phosphorylable K-Ras mutant exhibits a less sustained activation profile and do not efficiently activate AKT at low growth factor doses compared with wild-type K-Ras. It is interesting that the physiological responses induced by K-Ras are affected by this phosphorylation; when K-Ras cannot be phosphorylated it exhibits a remarkably decreased ability to stimulate proliferation in non-saturated serum conditions. Finally, we demonstrate that phosphorylation also regulates oncogenic K-Ras functions, as focus formation capacity, mobility and apoptosis resistance upon adriamycin treatment of cells expressing oncogenic K-Ras that cannot be phosphorylated are highly compromised. Moreover, at low serum concentration proliferation and survival is practically inhibited when cells cannot phosphorylate oncogenic K-Ras. In this condition, K-Ras phosphorylation is essential to ensure a proper activation of mitogen-activated protein kinase and PI3K/AKT pathways. In summary, our findings suggest that the interplay between CaM interaction and PKC phosphorylation is essential to regulate non-oncogenic and oncogenic K-Ras activity and functionality.
Alvarez-Moya
B
B
Departament de Biologia Cel.lular, Facultat de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain.
López-Alcalá
C
C
Drosten
M
M
Bachs
O
O
Agell
N
N
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000502:physiology*; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011493:Protein Kinase C / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002147:Calmodulin; D012694:Serine; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate