Identification of Essential Interacting Elements in K-Ras/Calmodulin Binding and Its Role in K-Ras Localization

Abstract

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 ␣-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؉

Oncogenic K-Ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

“…In fact, certain groups have reported that phosphomimetic K-Ras is internalized to intracellular membranes, such as those in mitochondria, the Golgi complex, the endoplasmic reticulum or in endosomes (Bivona et al, 2006;Fivaz and Meyer, 2005). However, in agreement with previous observations (Lopez-Alcalá et al, 2008;Plowman et al, 2008), we show here that, after simultaneous transfection of YFP-K-RasG12V-S181A and mCherry-K-RasG12V-S181D into HEK293 cells, both phospho-mutants are mainly located at the plasma membrane ( Fig. 2A).…”

K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function

“…This regulatory mechanism has already been shown for other CaM-binding proteins, such as neurogranin, neuromodulin, myristylated alanine-rich C-kinase substrate (Chakravarthy et al, 1999) and p21 (Rodriguez-Vilarrupla et al, 2005;Agell et al, 2006). In analogy to other CaM-binding proteins, phosphorylation within the CaM-binding region of K-Ras inhibits its binding to CaM (Lopez-Alcala et al, 2008). Thus, also for K-Ras, there is an antagonism between CaM and PKC: CaM inhibits phosphorylation by PKC and phosphorylation inhibits binding of CaM.…”

“…We have shown that CaM inhibition specifically enhances K-Ras activation provided protein kinase C (PKC) is active. It is interesting that we have also demonstrated that K-Ras is a CaM-binding protein, although it is only able to bind CaM if it is GTP-bound and not phosphorylated at Ser181, suggesting an interplay between CaM binding to K-Ras, K-Ras phosphorylation and K-Ras activity (Villalonga et al, 2001Lopez-Alcala et al, 2008).…”

“…It is interesting that HA-KRasG12V, transiently expressed in HEK293 cells, was also phosphorylated in the presence of growth factors, but this modification was dramatically reduced when CaM was simultaneously overexpressed. Finally, HA-KRasG12VDKK, a mutant with two Lys of the polybasic domain deleted that exhibits a reduced affinity for CaM (Lopez-Alcala et al, 2008), was still phosphorylated after CaM over-expression (Figure 1d). Consequently, we can conclude that K-Ras is phosphorylated in vivo in the presence of growth factors, or upon PKC stimulation and that this phosphorylation is inhibited by CaM interaction.…”

“…RBD (Ras-binding domain of Raf-1) pull downs were performed as previously described (Lopez-Alcala et al, 2008) to determine the amount of active Ras.…”