A substrate of ecto-protein kinase is microtubule-associated protein 1B in cortical cell cultures undergoing synaptogenesis.
Synapse formation between cultured rat cortical neurons is inhibited by the continuous application of K-252b, an ecto-protein kinase inhibitor, which cannot permeate the cell membrane. In order to identify the phosphorylated membrane proteins which are necessary for synapse formation, endogenous substrates for ecto-protein kinase activity were investigated. To detect phosphorylation of proteins containing extracellular domains, [gamma-33P]ATP was applied to the medium for brief periods. Proteins were then separated by SDS polyacrylamide gel electrophoresis and detected by autoradiography. Some bands showed immediate phosphorylation and this phosphorylation was suppressed by the addition of K-252b to the medium. We examined partial amino acid sequences of these substrates. The band with the highest molecular weight, whose phosphorylation was strongly inhibited by K-252b, was identified as microtubule-associated protein (MAP) 1B. These results suggest the possibility that the phosphorylation of extracellular domains of MAP1B is involved in synaptogenesis between cortical neurons.
Muramoto
K
K
Department of Molecular & Cellular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Japan.
Taniguchi
H
H
Kawahara
M
M
Kobayashi
K
K
Nonomura
Y
Y
Kuroda
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005333:Fetus; D026121:Indole Alkaloids; D013058:Mass Spectrometry; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009474:Neurons / Q000201:enzymology / Q000502:physiology*; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D013569:Synapses / Q000502:physiology*
D002227:Carbazoles; D026121:Indole Alkaloids; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; C049985:staurosporine aglycone; D011494:Protein Kinases; C034454:ectoprotein kinase; D011493:Protein Kinase C