Rapid T cell receptor-mediated SHP-1 S591 phosphorylation regulates SHP-1 cellular localization and phosphatase activity.
Since the tyrosine phosphatase SHP-1 plays a major role in regulating T cell signaling, we investigated regulation thereof by Ser/Thr phosphorylation. We found that T cell receptor (TCR) stimulation induced fast (<or=1 min) and transient phosphorylation of SHP-1 S591 in both Jurkat and human peripheral blood T-cells (PBT). Phosphorylation of S591 in T-cells could be mediated artificially by a constitutive active PKC-theta construct, but the dose dependence of inhibition by PKC inhibitors indicated that PKCs were not the relevant basophilic kinase in the physiological response. S591 phosphorylation inhibited phosphatase function since a S591D mutant had lower activity than the S591A mutant. Additional evidence that S591 phosphorylation alters SHP-1 function was provided by studies of Jurkat cells stably expressing SHP-1 wild type or mutants. In those cells, S591D mutation reduced the capacity of transfected SHP-1 to inhibit TCR-induced phosphorylation of PLC-gamma1. Interestingly, SHP-1 Y536 phosphorylation (previously shown to augment phosphatase activity) was also induced in PBT by TCR signal but at a much later time compared with S591 ( approximately 30 min). S591 phosphorylation also altered cellular distribution of SHP-1 because: 1) SHP-1 in lipid rafts and a sheared membrane fraction was hypophosphorylated; 2) In stably transfected Jurkat cell lines, S591D mutant protein had reduced presence in both lipid raft and the sheared membrane fraction; 3) S591 phosphorylation prevented nuclear localization of a C-terminal GFP tagged SHP-1 construct. Our studies also shed light on an additional mechanism regulating SHP-1 nuclear localization, namely conformational autoinhibition. These findings highlight elegant regulation of SHP-1 by sequential phosphorylation of serine then tyrosine.
Liu
Yin
Y
Experimental Immunology Branch, Bldg. 10/4B05 National Cancer Institute, NIH Bethesda, MD 20892, USA.
Kruhlak
Michael J
MJ
Hao
Jian-Jiang
JJ
Shaw
Stephen
S
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells / Q000378:metabolism; D021962:Membrane Microdomains / Q000201:enzymology / Q000235:genetics; D051379:Mice; D009154:Mutation; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D021381:Protein Transport; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism
D011948:Receptors, Antigen, T-Cell; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D012694:Serine; D011493:Protein Kinase C; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D051966:Phospholipase C gamma