H89 sensitive kinase regulates the translocation of Sar1 onto the ER membrane through phosphorylation of ER-coupled β-tubulin.
ER-to-Golgi protein transport is carried out by transport vesicles which are formed at the ER-exit sites with recruitment of cytoplasmic coat proteins. Vesicle formation is initiated by assembly of the small G protein (Sar1) onto the ER membrane. Sar1 assembly onto the ER membrane is suppressed by protein kinase inhibitor H89, suggesting participation of H89-sensitive kinase in this process. The present study identified an effector of H89-sensitive kinase by LC-MS PMF analysis combined with 1D- and 2D-PAGE autoradiography, and examined the changes on the effector and Sar1 translocation induced by H89. H89 significantly suppressed the phosphorylation of 55 kDa protein with dosage dependency, and phosphorylation of 55 kDa, pI 5.5 protein spot in 2-D-autoradiography was drastically diminished by H89. LC-MS PMF analysis showed that the protein spot was β-tubulin. H89 significantly suppressed Sar1 translocation onto the ER. These findings indicate that β-tubulin is one of downstream effectors of H89-sensitive kinase, and that suppression of ER-coupled β-tubulin phosphorylation decreases Sar1 translocation onto the ER, suggesting that phosphorylation of β-tubulin regulates Sar1 translocation.
Laboratory of Toxicology, Course of Veterinary Science, Graduate School of Life and Environmental Biosciences, Osaka Prefecture University, Izumisano, Japan.
Hiroshi
H
Nakagawa
Shuichi
S
Miyazaki
Takuto
T
Abe
Haruka
H
Umadome
Katsuhiro
K
Tanaka
Kazuhiko
K
Nishimura
Masayuki
M
Komori
Saburo
S
Matsuo
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology*; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D007546:Isoquinolines; D011994:Recombinant Proteins; D013449:Sulfonamides; D014404:Tubulin; D011494:Protein Kinases; C487879:SAR1 protein, rat; D020559:Monomeric GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide