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

Luc A. Sabourin,Paul O'Reilly,Ziad Chaar,Irwin Gelman

doi:10.1074/jbc.m605665200

Luc A. Sabourin, Paul O'Reilly + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m605665200

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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.

Highlights

C-Src and its viral counterpart v-Src are the most studied members of Src family kinases
Our results show that SLK kinase activity is reduced in cells expressing an oncogenic form of c-Src and that this regulation requires Src kinase activity and translocation to the cell periphery
This down-regulation does not proceed through focal adhesion kinase (FAK) or by direct tyrosine phosphorylation of SLK but rather through activation of CK2, which in turn down-regulates SLK

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Immunostaining—HEK293, COS1, and 49F cells were purchased from the American Tissue Type Collection. Prior to SLK kinase assays, cells stably expressing fpgv-1 or tsLA29 v-Src (maintained at 40 °C) were shifted to 35 °C for 24 – 48 h. The v-Src cDNA and mutant PCR products were subcloned into an HA-tagged pcDNA3 expression vector (Invitrogen) using standard cloning procedures [16]. To assay CK2 activity, 400 ␮g of total cell lysate was immunoprecipitated using 2 ␮g of anti-CK2␣ goat polyclonal antibodies (C-18; Santa Cruz Biotechnology) and washed three times with NETN and once with modified CK2 kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MnCl2, 10 mM NaF, 10 mM ␤-glycerophosphate, and 1 mM orthovanadate) [13]. 40 ␮g of total cell lysates was subjected to Western blot with anti-CK2␣ goat polyclonal antibodies (C-18; Santa Cruz Biotechnology) to detect endogenous CK2␣ proteins.

RESULTS

We performed SLK in vitro kinase assays on cell lysates from

Findings

DISCUSSION