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) induces a short-lived phosphorylation and activation of stress-activated protein kinase (SAPK) and cellular differentiation. To investigate whether the rapid 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 loop to control SAPK activity. Using both stable cell lines and transient transfection we demonstrate that activation of SAPK rapidly stimulated phosphorylation of M3/6. This phosphorylation did not regulate the half-life of total cellular M3/6. hVH5 and M3/6 shares with all sequenced mammalian DSPs an 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 this sequence or mutation of the LYL portion blocks the ability of this phosphatase to dephosphorylate SAPK.
Department of Medical Oncology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
T R
TR
Johnson
J R
JR
Biggs
S E
SE
Winbourn
A S
AS
Kraft
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D004789:Enzyme Activation / Q000187:drug effects; D005246:Feedback; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006207:Half-Life; D006801:Humans; D020014:K562 Cells; D051379:Mice; D048057:Mitogen-Activated Protein Kinase 10; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D048057:Mitogen-Activated Protein Kinase 10; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D017027:Protein Tyrosine Phosphatases; D013755:Tetradecanoylphorbol Acetate