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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.

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein that can regulate actin reorganization during cell adhesion and spreading (Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002) J. Biol. Chem. 277, 37685-37692). Because of its association with the microtubule network, we investigated whether SLK plays a role in cell cycle progression, a process that requires microtubule dynamics during mitosis. Consistent with microtubule association in exponentially growing cells, our results showed that SLK co-localizes with the mitotic spindle in cells undergoing mitosis. Expression of a kinase-inactive mutant or SLK small interfering RNAs inhibited cell proliferation and resulted in an accumulation of quiescent cells stimulated to re-enter the cell cycle in the G2 phase. Cultures expressing the mutant SLK displayed a normal pattern of cyclin D, E, and B expression but failed to down-regulate cyclin A levels, suggesting that they cannot proceed through M phase. In addition, these cultures displayed low levels of both phospho-H3 and active p34/cdc2 kinase. Overexpression of active SLK resulted in ectopic spindle assembly and the induction of cell cycle re-entry of Xenopus oocytes, suggesting that SLK is required for progression through G2 upstream of H1 kinase activation.

Cytoskeletal remodeling events are tightly regulated by signal transduction systems that impinge on adhesion components and modulators of cellular architecture. We have previously shown that the Ste20-like kinase (SLK) can induce apoptosis through the induction of actin disassembly and cellular retraction (Sabourin, L. A., Tamai, K., Seale, P., Wagner, J., and Rudnicki, M. A. (2000) Mol. Cell. Biol. 20, 684-696). Using immunofluorescence studies, we report that SLK is redistributed with adhesion components at large podosome-like adhesion sites in fibronectin-stimulated fibroblasts. However, in vitro kinase assays demonstrate that its activity is not modulated following fibronectin stimulation. Double immunofluorescence studies in exponentially growing or spreading fibroblasts show that SLK is associated with the microtubule network and can be coprecipitated with alpha-tubulin. Furthermore, the stimulation of adhesion site formation by microtubule-disrupting agents induces the relocalization of SLK with unpolymerized alpha-tubulin to large vinculin-containing adhesion complexes. Using microinjection studies, we show that ectopic expression of activated SLK induces the disassembly of actin stress fibers, a process that can be inhibited by dominant negative Rac1. Significantly, endogenous SLK can be colocalized with Rac1 in spreading cells on FN. Finally, the overexpression of SLK by adenoviral infection inhibits cell spreading on fibronectin. These results suggest that SLK is part of a microtubule-associated complex that is targeted to adhesion sites and implicated in the regulation of cytoskeletal dynamics.

Myotonic dystrophy is the most common inherited adult neuromuscular disorder with a global frequency of 1/8000. The genetic defect is an expanding CTG trinucleotide repeat in the 3'-untranslated region of the myotonic dystrophy protein kinase gene. We present the in vitro characterization of cis regulatory elements controlling transcription of the myotonic dystrophy protein kinase gene in myoblasts and fibroblasts. The region 5' to the initiating ATG contains no consensus TATA or CCAAT box. We have mapped two transcriptional start sites by primer extension. Deletion constructs from this region fused to the bacterial chloramphenicol acetyltransferase reporter gene revealed only subtle muscle specific cis elements. The strongest promoter activity mapped to a 189-base pair fragment. This sequence contains a conserved GC box to which the transcription factor Sp1 binds. Reporter gene constructs containing a 2-kilobase pair first intron fragment of the myotonic dystrophy protein kinase gene enhances reporter activity up to 6-fold in the human rhabdomyosarcoma myoblast cell line TE32 but not in NIH 3T3 fibroblasts. Co-transfection of a MyoD expression vector with reporter constructs containing the first intron into 10 T1/2 fibroblasts resulted in a 10-20-fold enhancement of expression. Deletion analysis of four E-box elements within the first intron reveal that these elements contribute to enhancer activity similarly in TE32 myoblasts and 10 T1/2 fibroblasts. These data suggest that E-boxes within the myotonic dystrophy protein kinase first intron mediate interactions with upstream promoter elements to up-regulate transcription of this gene in myoblasts.

The genetic defect underlying myotonic dystrophy (DM) has been identified as an unstable CTG trinucleotide repeat amplification in the 3'-untranslated region (3'-UTR) of the DM kinase gene (DMK). Individuals with the most severe congenital form display a marked delay in muscle terminal differentiation. To gain insight into the role of DMK during myogenesis, we have examined the effect of DMK overexpression on the terminal differentiation of the murine myoblast cell line C2C12. We demonstrate that a 4-10-fold constitutive overexpression of DMK mRNA in myoblasts caused a marked inhibition of terminal differentiation. Surprisingly, this activity was mapped to a 239-nucleotide region of the 3'-UTR of the DMK transcript. When the DMK 3'-UTR was placed downstream of a reporter gene, the same inhibition of myogenesis was observed. Following the induction of differentiation of myoblast clones overexpressing the DMK 3'-UTR, the levels of myogenin mRNA were reduced by approximately 4-fold, whereas the steady state levels of mef-2c transcripts were not affected. These data suggest that overexpression of the DMK 3'-UTR may interfere with the expression of musclespecific mRNAs leading to a delay in terminal differentiation.

Myotonic dystrophy is caused by the expansion of a CTG repeat found in the 3'-untranslated region of the myotonic dystrophy kinase. The mechanism of disease and the role of the kinase are currently obscure. Here we begin the investigation of domain structure/function correlations to aid in determining its normal function. Expressed full-length protein and protein truncated before a C-terminal hydrophobic domain were compared. In vitro, signal peptide function and protection of kinase by microsomal membranes were absent; thus, it is not translocated, as previously proposed. However, full-length kinase expressed in insect cells was found in fractions enriched for membranes and decorated mitochondria. The truncated form was found primarily in the cytosol. The kinase was present as two self-associated, disulfide-linked complexes. The majority of full-length kinase was found in the larger of the two complexes, while almost all of the truncated form was found in the smaller. Thus, the C-terminal region confers a higher order of self-association. Furthermore, full-length kinase expressed in COS-1 cells was present as high molecular weight complex, while the truncated form was present as monomer species. These experiments indicate that the myotonic dystrophy kinase is not membrane-integrated, but that it may have a molecular organization which favors peripheral association with membranes.