Detection and characterization of a rat parotid gland protein kinase that catalyzes phosphorylation of matured destrin at Ser-2.
Destrin, an actin-binding protein, is partly phosphorylated at Ser-2 (numbering on the matured form) in the resting rat parotid gland, and beta-adrenergic or cholinergic stimulation of this gland induces its dephosphorylation. In this study, we searched for the protein kinase involved in phosphorylation of destrin. We developed an assay method for the kinase, using an antibody specific to destrin phosphorylated at Ser-2, and detected the kinase in the rat parotid homogenate. This enzyme was predominantly (93%) present in the soluble fraction, and the enzyme in this fraction was characterized. It had an optimum pH at 6.8 and required 3-5 mM Mg2+ for its maximum activity. Ca2+ (1 mM) had no effect whereas Mn2+ (5 mM) inhibited the enzyme activity by 75%. The apparent Km values for destrin and ATP were 92 microg/ml and 170 microM, respectively. GTP was an inefficient phosphate donor, and cAMP had no effect. Heat-denatured destrin was poorly phosphorylated. Two-dimensional PAGE analysis of destrin phosphorylated with the soluble fraction indicated that it was exclusively phosphorylated at Ser-2. None of the protein kinase inhibitors tested here was specific to this enzyme. At 1 mM, ML-7, Y-27632, KN-93, HA-1077, H-7, and H-8 inhibited the activity by 88, 61, 58, 49, 46, and 42%, respectively. Staurosporine (2 microM) and H-89 (50 microM) inhibited the activity by 48 and 33%, respectively. Heparin (30 microg/ml) had no effect. These results suggest that the rat parotid gland contains a novel, constitutively active, soluble protein kinase catalyzing specific phosphorylation of destrin at Ser-2.
Nakano
Seigo
S
Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan.
Kanamori
Takao
T
Suzuki
Masami
M
Titani
Koiti
K
D051339:Actin Depolymerizing Factors; D000818:Animals; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003598:Cytoskeletal Proteins; D051345:Destrin; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008297:Male; D008840:Microfilament Proteins / Q000276:immunology; D010306:Parotid Gland / Q000201:enzymology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D051339:Actin Depolymerizing Factors; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D051345:Destrin; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D047428:Protein Kinase Inhibitors; D012694:Serine; D011494:Protein Kinases