K-Ras Binds Calmodulin-Related Centrin1 with Potential Implications for K-Ras Driven Cancer Cell Stemness

SIMPLE SUMMARY: Trafficking chaperones facilitate the spatio-temporal distribution pattern of proteins inside cells. In the case of the membrane-anchored protein Ras, trafficking chaperones typically bind to the C-terminal farnesyl-moiety. Thus shielded from the aqueous environment, Ras can diffuse more efficiently through the cytoplasm. The calcium-binding protein calmodulin (CaM) was proposed as a K-Ras trafficking chaperone. However, CaM has many different functions inside the cell. Centrin proteins are highly related to calmodulin, and we find that they also bind to K-Ras. Unexpectedly, this interaction depends on the activation state and the effector binding site of K-Ras, not on the farnesyl-anchor. Overall, CaM and centrin1 appear to enable only a fraction of K-Ras membrane anchorage. Given that CaM inhibitors also affect the K-Ras/centrin1 interaction and the very similar distribution of centrin1 and CaM throughout the cell cycle, the dependence of K-Ras on either protein may be difficult to determine. ABSTRACT: Recent data suggest that K-Ras4B (hereafter K-Ras) can drive cancer cell stemness via calmodulin (CaM)-dependent, non-canonical Wnt-signalling. Here we examined whether another Ca(2+)-binding protein, the CaM-related centrin1, binds to K-Ras and could mediate some K-Ras functions that were previously ascribed to CaM. While CaM and centrin1 appear to distinguish between peptides that were derived from their classical targets, they both bind to K-Ras in cells. Cellular BRET- and immunoprecipitation data suggest that CaM engages more with K-Ras than centrin1 and that the interaction with the C-terminal membrane anchor of K-Ras is sufficient for this. Surprisingly, binding of neither K-Ras nor its membrane anchor alone to CaM or centrin1 is sensitive to inhibition of prenylation. In support of an involvement of the G-domain of K-Ras in cellular complexes with these Ca(2+)-binding proteins, we find that oncogenic K-RasG12V displays increased engagement with both CaM and centrin1. This is abrogated by addition of the D38A effector-site mutation, suggesting that K-RasG12V is held together with CaM or centrin1 in complexes with effectors. When treated with CaM inhibitors, the BRET-interaction of K-RasG12V with centrin1 was also disrupted in the low micromolar range, comparable to that with CaM. While CaM predominates in regulating functional membrane anchorage of K-Ras, it has a very similar co-distribution with centrin1 on mitotic organelles. Given these results, a significant overlap of the CaM- and centrin1-dependent functions of K-Ras is suggested.