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 regulting T cell signaling, we investigated regulation thereof by Ser/Thr phosphorylation. We found that T cell receptor (TCR) stimulation induced fast (≤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‐γ1. 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 (∼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.