The b-lymphoid protein tyrosine phosphatase ptprot modulates b-cell activation following bcr engagement

Ricardo C.T. Aguiar, Katherine D. Kreinbrink, Margaret A. Shipp

Research output: Contribution to journalArticlepeer-review


B-cell antigen receptor (BCR) engagement results in the activation of several protein tyrosine kinases (PTKs), including Lyn, Fyn, Blk, Btk and Syk, and the subsequent tyrosine phosphorylation and activation of many critical signaling pathways. The degree of phosphorylation of these intracellular proteins determines B-cell fate (clonal expansion, differentiation or cell death) and is strictly regulated by the opposing activities of the PTKs and the protein tyrosine phosphatases (PTPs). We have recently identified a developmentally-regulated B-cell specific PTP, PTPROt, and showed that its overexpression promotes cell-cycle arrest in a DLB-CL model (Blood 94:2403, 1999). More recently, we found that endogenous PTPROt was upregulated upon BCR crosslinking but not via CD40 ligation. These data suggested a possible role for PTPROt in Bcell regulation following BCR engagement. To assess this possibility, we established a catalytically inactive (C325S) PTPROt transfectant in a DLB-CL cell line and compared patterns of phosphorylation in the PTPROt C325S mutant, PTPROt wild-type sense, PTPROt antisense and vector-only stable clones. Cell lysates from resting or anti-Igactivated transfectants were size-fractionated, blotted and probed with an anti-PTyr antibody. Following BCR engagement, there was a dramatic increase in the tyrosine phosphorylation of several target proteins in the PTPROt antisense and C325S mutant clones. These changes were most striking in proteins of - 53/56 kD, 70 kD and 120 kD. The p53/56 doublet was also hyperphosphorylated in non-Ig-activated PTPROt antisense and C325S mutant clones. We re-probed the filters with an anti-Lyn antibody and confirmed that the p53/p56 protein corresponded to PTK Lyn. To determine whether PTPROt directly affected Lyn phosphorylation, we co-transfected COS cells with Lyn and either PTPROt sense or PTPROt C325S. Cell lysates were immunoprecipitated with anti-Lyn and subsequently immunoblotted with anti-PTyr. In these experiments, the levels of phosphorylated Lyn were significantly higher in COS cells co-transfected with PTPROt C325S mutant rather than PTPROt sense (71% vs. 28% of the total Lyn, respectively). Taken together, these studies implicate PTPROt as a negative regulator of BCR signaling, possibly via modulation of Lyn phosphorylation. Disruption of PTPROt is likely to determine deregulated B-cell proliferation and might play a role in auto-immunity and cancer.

Original languageEnglish (US)
Pages (from-to)237a
Issue number11 PART I
StatePublished - Dec 1 2000
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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