Staurosporine-inhibitable protein kinase activity associated with secretory granule membranes isolated from rat submandibular gland cells.
Protein kinases, such as protein kinase C, have been shown to be associated with secretory granules and to regulate the event of exocytosis in various tissues including parotid salivary acinar cells. However, in submandibular acinar cells that play an important role in the secretion of proteins into the oral cavity, kinase activity on the granule membrane has not been explored. Therefore, in the present study, we isolated the secretory granules from rat submandibular acinar cells and investigated the localisation of protein kinases on the granule membrane. Initially, we isolated and purified secretory granules from rat submandibular acinar cells. Addition of [gamma-32P] ATP to granule-membrane lysate phosphorylated the granule-membrane-associated 26, 32, 55 and 58kDa proteins, suggesting the presence of endogenous kinase activity on the membrane. Moreover, the phosphorylation of 26 and 32kDa proteins was inhibited by staurosporine and K252a, both non-specific protein kinase C inhibitors. However, the phosphorylation of 26 and 32kDa proteins was not inhibited by other protein kinase C inhibitors, such as calphostin C, GF109203X and chelerythrine, indicating that protein kinase C was not responsible for the phosphorylation. In addition, H-89, ML-9, KN-62 and genistein did not appear to inhibit this phosphorylation, indicating that protein kinase A, myosin light chain kinase (MLCK), Ca2+/calmodulin-dependent protein kinase II (CAMKII) and tyrosine kinase were not involved in the phosphorylation of 26 and 32kDa proteins. Moreover, Ca2+ had no effect on the kinase activity. Therefore, our results suggest that an unidentified, staurosporine-inhibitable protein kinase activity is associated with the secretory granule membrane of rat submandibular acinar cells.
Seo
Su Ryeon
SR
Department of Oral Biology & Oral Science Research Centre, BK21 Project for Medical Sciences, Yonsei University College of Dentistry, Seoul, South Korea.
Kim
Yeun Ju
YJ
Moon
Seok Jun
SJ
Sugiya
Hiroshi
H
Shin
Dong Min
DM
Lee
Syng-Ill
SI
Seo
Jeong Taeg
JT
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D026121:Indole Alkaloids; D008565:Membrane Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000201:enzymology* / Q000648:ultrastructure; D019311:Staurosporine / Q000494:pharmacology*; D013363:Submandibular Gland / Q000201:enzymology*
D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; C049985:staurosporine aglycone; D011494:Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; D002118:Calcium