title: Enzymatic activity of protein kinase LOSK: possible regulatory role of the structural domain.
abstract: 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.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia. skel@genebee.msu.su
authors.forename: E S
authors.initials: ES
authors.lastname: Potekhina
authors.forename: L A
authors.initials: LA
authors.lastname: Zinovkina
authors.forename: E S
authors.initials: ES
authors.lastname: Nadezhdina
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004926:Escherichia coli / Q000378:metabolism; D000081410:Germinal Center Kinases; D006493:Heparin / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D004676:Myelin Basic Protein / Q000378:metabolism; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D011233:Precipitin Tests; D011479:Protamines / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000081410:Germinal Center Kinases; D006657:Histones; D004676:Myelin Basic Protein; D011479:Protamines; D011994:Recombinant Proteins; D011107:Polylysine; D000255:Adenosine Triphosphate; D006493:Heparin; D017346:Protein Serine-Threonine Kinases