multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid.When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.

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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.

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