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.
Johnson
T R
TR
Department of Medical Oncology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Biggs
J R
JR
Winbourn
S E
SE
Kraft
A S
AS
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