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

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)(2)(3)(4)(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 char-acterizing 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 Ste20like kinase SLK is redistributed to membrane ruffles and lamellipodia along with the microtubule network and adhesion components during cell spreading on fibronectin. In addition, SLK can induce actin stress fiber disassembly, which can be inhibited by inactive Rac1 (14), suggesting that SLK plays a role in cytoskeletal reorganization.
To gain further insights into the role of SLK in this process, we investigated whether regulators of early adhesion signaling events impact on SLK activity. 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. Furthermore, expression of v-Src in focal adhesion kinase (FAK)-null cells also resulted in SLK down-regulation, suggesting that v-Src-mediated SLK regulation is independent of FAK. Phosphoamino acid analysis of SLK in v-Src-expressing cells shows that SLK is hyperphosphorylated on serine residues and that v-Src-dependent SLK regulation is mediated, in part, by direct phosphorylation by CK2 in the SLK kinase domain. Our results suggest that CK2 may be regulating SLK at sites of cytoskeletal remodeling.

EXPERIMENTAL PROCEDURES
Cell Lines and Immunostaining-HEK293, COS1, and 49F cells were purchased from the American Tissue Type Collection. The mesodermal FAK Ϫ/Ϫ cells (p53 Ϫ/Ϫ , FAK Ϫ/Ϫ ), and wild-type counterpart were kindly provided by D. Ilic. The SYF cells, deficient for src, yes, and fyn and SYF cells stably expressing c-src were a generous gift from P. Soriano. All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco's modified Eagle's medium (Bio-Whitaker) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 g/ml penicillin, and 50 g/ml streptomycin.
For immunostaining the coverslips were fixed in 4% paraformaldehyde for 10 min at room temperature, washed in phosphate-buffered saline, and blocked with 1% bovine serum albumin or 200 g/ml ChromaPure Goat IgG (Jackson Immunoresearch Laboratories). SLK was detected using an anti-SLK rabbit polyclonal antibody (15) in conjunction with fluorescein isothiocyanate-labeled secondary antibodies. CK2 and c-Src proteins were detected with an anti-CK2 goat polyclonal (C-18; Santa Cruz) and an anti-c-Src monoclonal (Sigma) in conjunction with TRITC-labeled secondary antibodies. TRITC-conjugated phalloidin was used to detect actin stress fibers. Samples were visualized on a Zeiss Axioskop100 epifluorescence microscope equipped with appropriate filters and photographed with a digital camera (Sony Corporation HBO50) using the Northern Eclipse software package.
Plasmids-The pBabepuro3 v-src encoding the avian v-Src was kindly provided by M. McMahon. HA-tagged pcDNA3 expression vectors bearing full-length FAK, FAK kinase dead (FAK-K454R), the FAK Y397F mutant FRNK, and the FRNK S1084A mutant were kindly provided by D. Schlaepfer. The fpgv-1 control vector and the tsLA29 v-src encoding the temperature-sensitive v-Src were generously provided by M. C. Frame.
The HA-v-Src kinase inactive point mutant was generated using PCR-based mutagenesis converting lysine 298 to a methionine generating v-SrcK298M. Similarly, a HA-v-Src myristoylation site mutant (HA-v-SrcG2A) construction was also generated. The v-Src cDNA and mutant PCR products were subcloned into an HA-tagged pcDNA3 expression vector (Invitrogen) using standard cloning procedures (16). All PCRgenerated mutants were subjected to sequencing analysis at the Ottawa Health Research Institute. Sequencing data were analyzed using EditSeq and MegAlign (DNAStar computer software).
For SLK kinase assays, 400 g of total cell lysate was immunoprecipitated using 2 g of anti-SLK or 9E10 antibodies and 20 l of protein A-Sepharose (Amersham Biosciences) for 4 h at 4°C. Immunoprecipitates were washed three times with NETN (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.1%. Nonidet P-40) and once with SLK kinase buffer (20 mM Tris-HCl, pH 7.5, 15 mM MgCl 2 , 10 mM NaF, 10 mM ␤-glycerophosphate, and 1 mM orthovanadate). Kinase reactions (20 l in kinase buffer) were initiated by the addition of 5 Ci of [␥-32 P]ATP. After a 30-min incubation at 30°C, reactions were terminated by the addition of 4ϫ SDS sample buffer, and 20-l aliquots were fractionated by 8% SDS-PAGE. Gels were transferred to PVDF membranes and exposed to x-ray film. PVDF membranes were then probed for SLK to evaluate the efficiency of the immunoprecipitation.
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). Reactions (20 l in kinase buffer) were initiated by the addition of 3 g of dephosphorylated casein (C4032; Sigma) and 5 Ci of [␥-32 P]ATP. Reactions (30 min/37°C) were terminated by the addition of 4ϫ SDS sample buffer, and 20-l aliquots were fractionated by a gradient gel 8 -12% SDS-PAGE. Gels were transferred to PVDF membranes, Ponceau stained, and then exposed to x-ray film. 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.
Recombinant CK2 Kinase Assays-To assay SLK phosphorylation by CK2, recombinant SLK mutants were used in kinase assays with purified CK2. SLK GST fusions on glutathione-agarose were washed with NETN and CK2 kinase buffer. Reactions were initiated by the addition of 2 l of recombinant CK2 from rat liver (C3460; Sigma) and ATP as above. After a 30-min incubation at 37°C, beads were washed seven times with NETN to remove any traces of recombinant CK2 and once with PBS.
For tryptic digests, beads were incubated in 50 mM NH 4 HCO 3 with 10 units/l of trypsin from bovine pancreas (T8802; Sigma) overnight at 37°C, followed by the addition of another 10 l of 1 unit/l of trypsin for 4 h at 37°C the next day. The reaction was terminated by adding 4ϫ SDS sample buffer, boiled for 3 min, and fractionated on a 20% Tricine-SDS-PAGE. Expression of the undigested GST fusions was confirmed by independent SDS-PAGE and Coomassie staining of the purified undigested proteins.
Phosphoamino Acid Analysis-On the day of the assay, exponentially growing cells were washed twice and incubated in phosphate-free Dulbecco's modified Eagle's medium (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO 2 for 2 h. [ 32 P]Orthophosphate (250 Ci/ml) was then added for an additional 4 h. After the labeling period, cells were washed three times with TBS (preincubated at 4°C) and lysed using modified radioimmune precipitation buffer with protease inhibitors as above. Equal amounts of cell lysates were used to immunoprecipitate endogenous SLK using 2 g of anti-SLK antibodies overnight at 4°C. SLK protein was eluted off the beads with 50 l of redistilled 5.7 M HCl and hydrolyzed at 110°C for 1 h. Dried samples were then dissolved in 2 l of pH 1.9 buffer (formic acid/ acetic acid) containing 1 mg/ml of xylene cyanol, phosphoserine, phosphothreonine, and phosphotyrosine standards (Sigma). Samples were subjected to thin layer chromatography in pH 1.9 buffer followed by electrophoresis in pH 3.5 buffer (pyridine/ acetic acid). Amino acid standards were visualized with 0.2% ninhydrin followed by exposure to x-ray film.

v-Src Down-regulates SLK Kinase
Activity-Fibronectin stimulation of fibroblasts has been shown to trigger the activation of FAK, the recruitment of c-Src, and the formation of adhesion signaling complexes mediating actin rearrangements (18,19). Furthermore, FAK and c-Src appear to be critical for the turnover of these complexes, allowing cell migration. We have previously shown that SLK can induce actin disassembly and that it is redistributed to membrane structures reminiscent of ruffles and lamellipodia during cell spreading on FN (14). Therefore, to gain further insights into the role of SLK in cytoskeletal remodeling, we have investigated SLK activity in FAK-and SYF-deficient cell lines or v-Src-transformed cells.
We performed SLK in vitro kinase assays on cell lysates from FAK-WT, FAK Ϫ/Ϫ , SYF-deficient, SYFϩc-src, and 49F fibroblasts stably expressing the v-src oncogene. In vitro kinase assays showed no change in SLK kinase activity in SYF-deficient, SYFϩc-src, FAK-null, or FAK-WT cells (Fig. 1A). However, a 2-to 3-fold decrease in SLK kinase activity was consistently observed in v-Src-transformed cells (Fig. 1), suggesting that activated c-Src or its downstream effectors negatively regulates SLK. In addition, 49F cells stably expressing c-SrcY527F, a constitutively active mutant of c-Src, also showed a downregulation of SLK kinase activity (not shown).
To further understand the mechanism by which v-Src downregulates SLK activity, we generated 49F cells stably expressing tsLA29v-Src, a mutant v-Src that is temperature sensitive for translocation to adhesion sites (20). Shifting the cultures to the permissive temperature (35°C) results in the translocation of v-Src to the cell periphery. Incubation of the cells at the permissive temperature resulted in a 2-to 3-fold decrease in SLK kinase activity concomitant with the characteristic v-Src-induced morphological changes (see Fig. 1, F-H). Supporting a role for c-Src in SLK regulation, immunofluorescence staining of exponentially growing 49F cells revealed that c-Src and SLK proteins could be co-localized at membrane ruffles and lamellipodia ( Fig. 1, C and D). Furthermore, shifting the tsLA29v-Src cells to the permissive temperature also resulted in SLK v-Src co-localization (Fig. 1, E and F). Interestingly, immunoprecipitation of SLK followed by immunoblotting for phosphotyrosine residues revealed that SLK is not tyrosine phosphorylated under these conditions (not shown). Taken together, these results suggest that v-Src-mediated SLK down-regulation is indirect and that this requires v-Src translocation to the membrane.

v-Src Kinase Activity and Membrane Translocation Are Required for SLK Down-regulation-Previous studies have
shown that c-Src kinase activity is required for Rac1-and cdc42-induced adhesion remodeling and directed cell migration whereas the Src SH3 and SH2 domains are sufficient for its translocation to the cell periphery in a RhoA-dependent manner (20,21). Therefore, we tested the role of v-Src kinase activity and myristoylation site on SLK down-regulation. A kinase inactive (v-SrcK298M) and myristoylation-defective (v-SrcG2A) mutant was engineered and co-transfected with Myc-SLK in HEK293 cells. SLK immunoprecipitation and in vitro kinase assays showed that both v-Src point mutants could no longer down-regulate SLK kinase activity compared with controls ( Fig. 2A). Together, these data suggest that both v-Src kinase activity and membrane anchoring are required to negatively regulate SLK kinase activity.
v-Src-mediated Down-regulation of SLK Kinase Activity Is Independent of FAK-Integrin signaling following FN stimulation proceeds through the recruitment and autophosphorylation of FAK on Tyr-397, causing its transient association with activated c-Src and further phosphorylation (4). Similarly, in v-Src-transformed cells, FAK is hyperphosphorylated, in addition to other adhesion complex proteins (22)(23)(24). Therefore, we investigated whether SLK could also be down-regulated by FAK. To test this, HEK293 cells were co-transfected with Myc-SLK and either wild-type FAK, alone or in combination with the dominant negative FRNK molecule, or the non-FAK binding mutant FRNK S1084A (25,26). In vitro kinase assays showed that FAK overexpression had no effect on SLK kinase activity (Fig. 2B). Similarly, overexpression of FRNK had no significant effect. Supporting these observations, SLK kinase activity was still down-regulated in FAK-WT or FAK Ϫ/Ϫ cells stably expressing the oncoprotein v-Src (Fig. 2C). Together, these results suggest that v-Src-mediated SLK down-regulation does not proceed through FAK and its downstream signaling pathways but rather by activating an independent signaling system. SLK Is Hyperphosphorylated in v-Src-transformed Cells-To investigate whether potential post-translational modifications on SLK, other than tyrosine phosphorylation, were responsible for the negative down-regulation, we conducted phosphoamino acid analysis. Endogenous SLK protein was immunoprecipitated from 32 P-labeled control or v-Src-transformed cells, subjected to acid hydrolysis, and resolved by two-dimensional thin layer chromatography. Phosphoamino acid analysis revealed that SLK is hyperphosphorylated solely on serine FIGURE 2. v-Src kinase activity and membrane localization are required for SLK regulation. A, HEK293 cells were co-transfected with HA-v-Src or mutants in the presence or absence of Myc-SLK. Myc-SLK was immunoprecipitated from cell lysates and subjected to in vitro kinase assay. The SLK immunoprecipitate was monitored by probing the kinase assays with anti-SLK antibodies. Expression of all mutants and SLK was confirmed by Western blotting of the total cell lysates for Myc and HA. B, v-Src-mediated SLK down-regulation is independent of FAK. HEK293 cells were co-transfected with Myc-SLK and FAK constructs and subjected to SLK kinase assays. Overexpression of FAK or FRNK had no effect on SLK kinase activity. SLK immunoprecipitates were monitored by reprobing the kinase assay for SLK protein. Expression of the various forms of FAK was verified by Western blotting (bottom panel). C, SLK was immunoprecipitated from FAK ϩ/ϩ or Ϫ/Ϫ mouse embryonic fibroblasts stably expressing v-Src and subjected to in vitro kinase assays. The immunoprecipitate was assessed as in panel A, and FAK expression was also evaluated by Western blot analysis. residues in v-Src-or c-SrcY527Ftransformed 49F cells in comparison to 49F cells (Fig. 3A), supporting the notion that the v-Src-or c-SrcY527F-mediated effect is indirect. Treatment of 49F cells with phosphatase inhibitors followed by SLK immunoprecipitation showed no changes in kinase activity (not shown), suggesting that v-Src-mediated SLK down-regulation proceeds through the modulation of a serine/threonine kinase. To identify potential kinases involved in the down-regulation of SLK activity, we have focused on amino acids 1-373, encompassing all of the kinase subdomains for the identification of target serine residues. To that end, Myc-tagged SLK kinase inactive constructs (Myc-SLK 1-373 K63R, Myc-SLK  K63R, and Myc-SLK 1-325 K63R), each designed to contain a small number of serine residues and encompassing the kinase domain, were generated. Co-transfection of these constructs with or without v-Src in HEK293 cells revealed that only the Myc-SLK 1-373 K63R truncation was hyperphosphorylated, suggesting a potential phosphorylation occurring within the last 9 serine residues of the kinase domain (Fig. 3B). The peptide sequence containing these last serine residues was analyzed for potential protein phosphorylation sites using ScanProsite from ExPASy. Primary sequence analysis showed the presence of putative protein kinase A, protein kinase C, and CK2 phosphorylation sites (Fig. 3C). To test the role of protein kinase A and protein kinase C on SLK kinase activity, HEK293 cells were transfected with HA-SLK and treated with either dibutyryl cyclic AMP or 12-O-tetradecanoylphorbol-13-acetate. Following SLK immunoprecipitation and kinase assays, no changes in SLK kinase activity were observed (not shown). Supporting this, Myc-tagged SLK point mutants S340A and S364A, sites of potential protein kinase A and protein kinase C phosphorylation, respectively, showed no changes in kinase activity at the basal level or when co-expressed with HA-v-Src (not shown), suggesting that the v-Src effect is not mediated by protein kinase A or protein kinase C.
CK2 Phosphorylates SLK and Regulates Its Kinase Activity-To investigate the potential role of CK2 on SLK kinase activity, Myc-SLK point mutants for serine residues 347/348 and 362 were generated (Myc-SLK SS347/348AA and Myc-SLK S362A). HEK293 cells transfected with these constructs revealed that Myc-SLK SS347/8AA displayed a 2-to 3-fold increase in basal kinase activity when compared with Myc-SLK (Fig. 4A). In addition, co-transfection of Myc-SLK SS347/ FIGURE 3. v-Src-dependent phosphorylation of the SLK kinase domain. A, phosphoamino acid analysis of endogenous SLK protein from v-Src-and c-SrcY527F-transformed cells. Cells were labeled with [ 32 P]orthophosphate, lysed, and subjected to SLK immunoprecipitation. The phosphorylated SLK was then hydrolyzed, spotted onto thin layer chromatography plates, and exposed to x-ray film. The position of the phosphorylated standards is circled. B, Myc-tagged SLK kinase inactive truncations SLK 1-192 K63R, SLK 1-325 K63R, and SL 1-373 K63R were co-transfected with v-Src, and the cells were labeled with [ 32 P]orthophosphate. SLK 1-373 K63R truncations were immunoprecipitated, transferred to polyvinylidene difluoride membranes, and exposed to x-ray film. Control transfections consisted of the empty expression vector (pCanHA2) and Myc-SLK constructs. The PVDF membranes were probed with an anti-Myc antibody to evaluate the efficiency of the immunoprecipitation (not shown). C, the peptide sequence comprised from SLK residue 332 to 370 illustrating the potential serines hyperphosphorylated in v-Src-transformed cells as well as kinase target sites. Myc-SLK mutants were transfected into HEK293 cells in the absence or presence of HA-v-Src, and 9E10 immunoprecipitates were subjected to in vitro kinase assays. Samples were transferred to PVDF membranes and exposed to x-ray film. The membranes were then probed with an anti-SLK antibody to evaluate the efficiency of the immunoprecipitations. Endogenous levels of CK2␣ expressed in HEK293 cells are also shown. B, equal amounts of cell lysates probed with 12CA5 show the efficiency of the HA-v-Src transfection.
348AA with HA-v-Src did not result in the down-regulation of SLK kinase activity (Fig. 4A). Examination of the CK2a protein content in HEK293 cells revealed that it is highly expressed (Fig.  4A), suggesting that CK2 may potentially regulate the catalytic activity of the transfected Myc-SLK (Fig. 4A). Therefore, we next addressed whether SLK is directly phosphorylated by CK2 on serine 347/348 or 362. The kinase domain mutants were GST tagged and subjected to an in vitro CK2 kinase assay in the presence or absence of recombinant rat liver CK2 (Sigma). Following the kinase assay, samples were either digested with thrombin to remove the GST peptide or with trypsin to reveal the 1.5-kDa peptide of interest containing serines 347/348 or 362 (Fig. 5A). Both thrombin and trypsin digests showed that the SLK mutant SS347/348AA displayed a marked decrease in its phosphorylation level relative to GST-SLK 1-373 K63R or GST-SLK 1-373 K63R S362A. These results suggest that CK2 can phosphorylate SLK directly on serine residues 347/348 and that this results in SLK down-regulation.
Inhibition of CK2 Rescues SLK Kinase Activity in v-Srctransformed Cells-To assess the role of CK2 on SLK kinase activity in v-Src-transformed cells, we used 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB) (Calbiochem), a specific CK2 inhibitor on v-Src-expressing cells (27). Control and v-Srctransformed 49F cells were first treated overnight with 50 mM TBB. SLK and CK2 were then immunoprecipitated independently from the same cell lysate and subjected to in vitro kinase assays. Our results show that after TBB treatment SLK kinase activity in v-Src-transformed cells is similar to that of wild-type 49F cells (Fig. 6A). Interestingly, we observed a dramatic increase in CK2 kinase activity in v-Srctransformed cells, suggesting that v-Src transformation results in CK2 activation (Fig. 6B). As previously reported, TBB treatment resulted in a decrease in CK2 kinase activity in both cell lines. Supporting a role for CK2 in the regulation of SLK activity, SLK and CK2␣ could be co-localized at the cell periphery in 49F cells following a 20-min replating assay on FN matrix (Fig. 6C). A, GSTtagged kinase inactive SLK (SLK 1-373 K63R) harboring mutations at serine residues 347/348 and 362 were generated (SLK-AA and SLK-A) and subjected to in vitro kinase assays in the presence or absence of rat liver recombinant CK2 (rCK2). Samples were digested with thrombin (A) or trypsin (B), resolved by SDS-PAGE, and exposed to x-ray film. The Coomassie-stained gel in panel A shows the GST fusions following thrombin treatment. The stained gel in panel B shows the GST fusions prior to trypsin digestion. FIGURE 6. Inhibition of CK2 restores SLK kinase activity in v-Src-transformed 49F cells. 49F and v-Src-transformed 49F cells were incubated with or without 50 mM TBB and SLK (A) as well as CK2␣ (B) were immunoprecipitated independently from the same cell lysate and subjected to in vitro kinase assays. SLK kinase activity was assayed based on its level of autophosphorylation (A) whereas dephosphorylated casein was used as a substrate for CK2 activity (B). Immunoprecipitates were probed with anti-SLK or with anti-CK2␣ antibodies to evaluate the efficiency of the immunoprecipitation. C, co-localization of SLK and CK2␣ during cell spreading on FN. 49F cells were fixed and stained at 20 min following replating of suspended cells onto FN-coated matrices. Co-localization of SLK and CK2 is observed in ruffles (arrowheads).

DISCUSSION
We have previously described that overexpression of SLK in various cell lines induces the rapid disassembly of actin stress fibers and cell death (28). Here we have shown that v-Src expression leads to down-regulation of SLK kinase activity, a process that requires v-Src translocation to the membrane, kinase activity, and membrane anchoring. Surprisingly, this down-regulation does not proceed through FAK or by direct tyrosine phosphorylation of SLK but rather through activation of CK2, which in turn down-regulates SLK. We have observed a marked increase in SLK phosphoserine content in v-Src-and c-SrcY527F-transformed 49F cells in comparison with parental cells. Further mapping identified two serine residues located at position 347/348 (SS347/348) that are phosphorylated by CK2. Although we have focused on the catalytic region of SLK as the target of v-Src-mediated down-regulation, truncation analysis of the full-length SLK revealed that SLK is also serine phosphorylated on its M-NAP and ATH domains (15,28), however at much lower levels (not shown). Therefore, serine phosphorylation of those domains cannot be excluded as other important regulatory sites. Nonetheless, serine 347/348 residues are the main CK2 targets within the kinase domain. Previous studies have shown that the Src family kinases Lyn and c-Fgr are capable of phosphorylating CK2␣ subunit, leading to an increase in its kinase activity in vitro (13). Interestingly, we have observed a marked increase in CK2 activity in v-Src-transformed cells that could not be attributed to differences in protein expression levels. Therefore, one possibility is that v-Src may directly modulate CK2 activity. Supporting this, we have demonstrated that the inhibition of CK2 activity in v-Src-transformed cells restores the catalytic activity of SLK.
We have previously demonstrated that SLK is redistributed with vinculin to structures reminiscent of membrane lamellipodia and ruffles during cell spreading on FN (14). Similarly, CK2 can be co-localized with SLK in these structures (Fig. 6). In addition, our previous results show that SLK-mediated actin stress fiber disassembly can be inhibited by the co-expression of a dominant negative Rac1 (RacN17). Interestingly, Timpson et al. (21) showed that c-Src kinase activity is required at peripheral adhesion sites for Rac1-and cdc42-induced adhesion remodeling and directed cell migration. Therefore, one possibility is that the regulation of cytoskeletal dynamics by SLK may be controlled by c-Src. Alternatively, the observed decrease in SLK kinase activity in v-Src-expressing cells may be specific to v-Src transformation.
A role for CK2 in cell survival has been demonstrated (8). In addition, SLK overexpression has been shown to induce an apoptotic response (15,28,29). Therefore, another possibility is that SLK activity is down-regulated in v-Src-transformed cells as part of an anti-apoptotic pathway. The signaling mechanisms that ensue downstream of SLK and how they regulate cytoskeletal remodeling and cell death will allow these hypotheses to be tested.