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Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-DeltaT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-DeltaT have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

LOSK (LOng Ste20-like Kinase) protein kinases of mammals belong to a recently identified family of GCK kinases which are involved in the induction of apoptosis. LOSK have an N-terminal acidic catalytic domain and a long C-terminal basic structural domain which is cleaved off in cells by caspases during apoptosis. To study the LOSK enzymatic activity and its dependence on the structural domain, two preparations of this protein kinase were prepared: a natural full-length protein immunoprecipitated from CHO-K1 cultured cells and a recombinant N-terminal catalytic fragment synthesized in E. coli. Both preparations displayed the ability for autophosphorylation and the ability for phosphorylation of MBP and of H1 histone, and their activities were comparable. H1 histone was a better substrate for LOSK than casein and ATP was a better substrate than other nucleotides. The pH dependence of the activity of the immunoprecipitated protein was more pronounced than the pH dependence of its recombinant fragment deprived of the C-terminal domain. The catalytic and the structural domains of LOSK can interact through electrostatic forces; therefore, effects were studied of various polyions at the concentration of 0.1 mg/ml on the activity. Heparin, protamine sulfate, and poly(L-Lys) decreased tenfold the ability of the full-length kinase to phosphorylate H1 histone. Heparin did not affect the activity of the recombinant fragment, whereas protamine sulfate and poly(L-Lys) had a slight effect. Moreover, protamine increased fourfold the autophosphorylation of the immunoprecipitated protein kinase. These data suggest that the structural C-terminal domain of LOSK should be involved in the regulation of its protein kinase activity: the LOSK protein kinase with C-terminal domain cleaved off could significantly less depend on conditions in the cell than the full-size enzyme.

Monoclonal antibody raised against a preparation of loach fish sperm centrosomes was used for screening of cDNA expressing library of Chinese hamster CHO-K1 cells. Two positive clones appeared to encode 628 amino acid protein fragment that was 72% identical to human KIAA0204 protein, i.e. putative protein kinase. Polyclonal antibodies raised against products of cDNA expression in E. coli recognized 210-kDa polypeptide in CHO-K1 cells and immunostained nuclear speckles, centrosomes and microtubules in these cells. The 210-kDa polypeptide (named MAK-L) co-sedimented with exogenous microtubules. Thus, one more protein kinase seems to be associated with the microtubule network in vertebrate cells.