Identification of essential interacting elements in K-Ras/calmodulin binding and its role in K-Ras localization.
We previously showed that K-Ras is a calmodulin-binding protein. Involvement of this interaction in anterograde and retrograde transport of K-Ras was then suggested. To test this we have analyzed here the domains of K-Ras essential for the interaction with calmodulin. At least three different regions in the K-Ras molecule were important; they are the hypervariable region, the alpha-helix between amino acids 151 and 166, and the Switch II. Within the hypervariable region, both the hydrophobic farnesyl group and the positive-charged amino acids were essential for the interaction between K-Ras and calmodulin in cellular extracts. Consistently, K-Ras S181D, which mimics phosphorylation of Ser-181 of K-Ras, also completely abolished binding to calmodulin. K-Ras mutants correctly farnesylated that did not bind calmodulin were all located at plasma membrane, showing that calmodulin interaction was not required for the transport of K-Ras to plasma membrane. In NIH3T3 cells, K-Ras and calmodulin colocalized mainly in the plasma membrane even after the addition of Ca(2+) ionophore, indicating that interaction did not directly lead to K-Ras internalization. Furthermore, using a K-Ras with impaired binding to calmodulin but with membrane localization, we could demonstrate in striatal neurones that interaction between K-Ras and calmodulin was not required for Golgi K-Ras translocation induced by Ca(2+) influx.
Lopez-Alcalá
Cristina
C
Departament de Biologia Cellular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.
Alvarez-Moya
Blanca
B
Villalonga
Priam
P
Calvo
Maria
M
Bachs
Oriol
O
Agell
Neus
N
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D011905:Genes, ras; D007476:Ionophores / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D020349:Surface Plasmon Resonance; D018631:ras Proteins / Q000378:metabolism*
D002147:Calmodulin; D007476:Ionophores; D018631:ras Proteins; D002118:Calcium