Identification of major Ca(2+)/calmodulin-dependent protein kinase phosphatase-binding proteins in brain: biochemical analysis of the interaction.
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH.
Ishida
Atsuhiko
A
Department of Biochemistry, Asahikawa Medical College, Asahikawa, Japan. aishida@asahikawa-med.ac.jp
Tada
Yukiyo
Y
Nimura
Takaki
T
Sueyoshi
Noriyuki
N
Katoh
Tsuyoshi
T
Takeuchi
Masayuki
M
Fujisawa
Hitoshi
H
Taniguchi
Takanobu
T
Kameshita
Isamu
I
D000818:Animals; D001921:Brain / Q000201:enzymology*; D003067:Coenzymes / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D000071636:Protein Phosphatase 2C; D051381:Rats
D003067:Coenzymes; D009479:Neuropeptides; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; D005634:Fructose-Bisphosphate Aldolase