v-Src-dependent Down-regulation of the Ste20-like Kinase SLK by Casein Kinase II

Abstract

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein inducing actin stress fiber disassembly. Here, we show that v-Src expression can down-regulate SLK activity. This down-regulation is independent of focal adhesion kinase but requires v-Src kinase activity and membrane translocation. SLK down-regulation by v-Src is indirect and is accompanied by SLK hyperphosphorylation on serine residues. Deletion analysis revealed that casein kinase II (CK2) sites at position 347/348 are critical for v-Src-dependent modulation of SLK activity. Further studies show that CK2 can directly phosphorylate SLK at these positions and that inhibition of CK2 in v-Src-transformed cells results in normal kinase activity. Finally, CK2 and SLK can be co-localized in fibroblasts spreading on fibronectin-coated substrates, suggesting a mechanism whereby SLK may be regulated at sites of actin remodeling, such as membrane lamellipodia and ruffles, through CK2.Cell growth and differentiation are tightly regulated mechanisms involving a large number of signaling cascades. Dysregulation and accumulation of genetic aberrations in these signaling cascades are key components in the transformation of a normal cell to a cancer cell. Furthermore, a direct correlation has been found between the metastatic potential of cancers and the nature of the observed genetic mutations (1-5). Indeed, cellular transformation by the src oncogene, a non-receptor tyrosine kinase, results in loss of adherence, invasiveness, and metastasis through increased phosphorylation of adhesion proteins and cytoskeletal disorganization.c-Src and its viral counterpart v-Src are the most studied members of Src family kinases. Several studies have illustrated potential Src-mediated mechanisms regulating cell survival and apoptosis (6). In addition, studies have demonstrated altered c-Src kinase activity, and in some cases protein levels, in human cancers such as breast, colon, and pancreatic cancers (3). v-Src-transformed cells have been widely used to elicit the oncogenic effect of a constitutively active c-Src. Features characterizing these cancer cell line models include increased cell detachment and migration.Casein kinase II (CK2) 3 is a serine/threonine kinase tetramer complex composed of two catalytic subunits, ␣ and/or ␣Ј and/or ␣Љ and two regulatory ␤ subunits. CK2 minimal amino acid consensus phosphorylation sequence is Ser-X-X-acidic, where the acidic residue can be glutamic acid, aspartic acid, phospho-Ser, or phospho-Tyr (7). CK2 is referred to as "a housekeeping enzyme," given its increasing number of substrates (Ͼ300). A role for CK2 has been shown in a wide range of cellular functions and properties such as cell proliferation, survival, differentiation, transformation, and tumorigenesis (8 -12). Recently, the CK2␣ subunit has been shown to be phosphorylated by the Src family kinases, c-Fgr and c-Lyn, resulting in increased catalytic activity (13).Previous studies in our laboratory have shown that the Ste20-like kinase SLK is redi...

Ste20-Related Kinase SLK Phosphorylates Ser188 of RhoA to Induce Vasodilation in Response to Angiotensin II Type 2 Receptor Activation

“…Serine phosphorylation of SLK at position 347/348 downregulates its kinase activity and the phosphorylation-resistant SS/AA-SLK mutant obtained by point mutation of these serine residues displayed a high kinase activity. 12 These in vitro and in-cell results thus suggest that AT 2 R-induced Ser188 phosphorylation of RhoA is mediated by SLK.…”

“…12 If CK2 is the kinase responsible for the basal phosphorylation of SLK in vascular smooth muscle cells, it is expected that inhibition of CK2 would lead to increase SLK kinase activity and, in turn, increase RhoA phosphorylation. The CK2 inhibitor 1 induced a 4-to 7-fold increase in RhoA phosphorylation in cells expressing SLK ( Figure 5B).…”

Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase

“…LOSK is indirectly slightly downregulated in vivo by overexpression of v-Src, probably via phosphorylation of its casein kinase II sites in the M1f fragment. However, activation of casein kinase II had no discernible effect on LOSK in vivo (Chaar et al, 2006). Cell polarization depends on Cdc42-regulated signal transduction pathway, which involves the activation of the Par6/ aPKC complex and inhibition of GSK-3beta (Schlessinger et al, 2007); it seems probable that LOSK participates in these events.…”

Ste20-like kinase SLK, at the crossroads

“…57 Interestingly, overexpression of v-src leads to inhibition of SLK activity through hyper-phosphorylation of the kinase domain through casein kinase II, suggesting that the src family kinases can also function to negatively regulate SLK activity during cell migration, perhaps during focal contact assembly. 59 Confirming a role for SLK in cell migration, knockdown experiments and expression of dominant negative SLK show marked reductions in migration. 57 Scratch wounding of confluent monolayers causes FAK activation as the cells migrate into the wound.…”

“…60 FA turnover during cell migration induces the formation of a functional FAK/ src complex initiated by auto-phosphorylation of FAK at tyrosine residue 397 (pY397), stable focal adhesion assembly and FA turnover and migration. [41][42][43][57][58][59][60] Similarly, nocodazole treatment of fibroblasts results in microtubule depolymerization and FA stabilization as evidenced by high levels of phospho-FAK-Y397. 61 Nocodazole wash-out and microtubule regrowth is accompanied by cyclical changes in the levels of FAK pY397.…”