Dephosphorylation of endogenous GABAB receptor R2 subunit and AMPK α subunits which were measured by in vitro method using transfer membrane

Protein phosphorylation can be regulated by changes in kinase activity, phosphatase activity, or both. GABAB receptor R2 subunit (GABABR2) is phosphorylated at S783 by 5′-AMP-activated-protein kinase (AMPK), and this phosphorylation modulates GABAB receptor desensitization. Since the GABAB receptor is an integral membrane protein, solubilizing GABABR2 is difficult. To circumvent this problem and to identify specific phosphatases that dephosphorylate S783, we employed an in vitro assay based on dephosphorylation of proteins on PVDF membranes by purified phosphatases. Our method allowed us to demonstrate that S783 in GABABR2 is directly dephosphorylated by PP2A (but not by PP1, PP2B nor PP2C) in a dose-dependent and okadaic acid-sensitive manner. We also show that the level of phosphorylation of the catalytic subunit of AMPK at T172 is reduced by PP1, PP2A and PP2C. Our data indicate that PP2A dephosphorylates GABABR2(S783) less efficiently than AMPK(T172), and that additional phosphatases might be involved in S783 dephosphorylation.

► A method for the dephosphorylation of endogenous proteins was approached.
► The method demonstrated the dephosphorylation in the concentration dependent manner.
► PP2A dephosphorylated specifically GABAB but less effectively than its upper AMPK.
► Okadaic acid increased both phosphorylation levels of GABAB and AMPK.
► With this method to find more effective phosphatases for them would be our next aim.