Regulation of dual-specificity phosphatases M3/6 and hVH5 by phorbol esters - Analysis of a delta-like domain

Treatment of leukemic cells with phorbol 12-myristate 13-acetate (PMA) indu ces a short-lived phosphorylation and activation of stress-activated protei n kinase (SAPK) and cellular differentiation. To investigate whether the ra pid deactivation of SAPK results from dephosphorylation by dual-specificity phosphatases (DSPs), we studied regulation of the DSP hVH5 and its murine orthologue M3/6 in K562 human leukemia cells. PMA treatment rapidly induced hVH5 transcripts in these cells, and induced expression of M3/6 completely inhibited PMA-stimulated phosphorylation of SAPK, suggesting a feedback lo op to control SAPK activity. Using both stable cell lines and transient tra nsfection we demonstrate that activation of SAPK rapidly stimulated phospho rylation of M3/6. This phosphorylation did not regulate the half-life of to tal cellular M3/6. hVH5 and M3/6 shares with all sequenced mammalian DSPs a n amino acid motif, XILPXLXL, located approximately 80 amino acids from the active site. The hVH5-M3/6 sequence, RILPHLYL, shares significant homology with the SAPK binding site of the c-Jun protein, called the delta domain. This motif was found to be important for DSP function, because deletion of RILPHLYL inhibits SAPK-mediated phosphorylation of M3/6, and deletion of th is sequence or mutation of the Ln portion blocks the ability of this phosph atase to dephosphorylate SAPK.