Abstract

The strength and duration of B-cell receptor (BCR) signaling depends upon the balance between protein tyrosine kinase (PTK) activation and protein tyrosine phosphatase (PTP) inhibition. BCR-dependent activation of the SYK PTK initiates downstream signaling events and amplifies the original BCR signal. Although BCR-associated SYK phosphorylation is clearly regulated by PTPs, SYK has not been identified as a direct PTP substrate. In an earlier screen for genes that might contribute to the pathogenesis of diffuse large B-cell lymphoma (DLBCL), we identified and preliminarily characterized a lymphoid PTP termed PTPROt (truncated). PTPROt is a member of the PTPRO family (also designated GLEPP, PTP-ϕ, PTP-oc and PTPu2), a group of highly conserved receptor-type PTPs that are thought to function as tumor suppressor genes. In earlier functional assays, lymphoma cells overexpressing PTPROt exhibited markedly increased G0/G1 arrest, providing the first functional evidence that this PTP regulated tumor growth. We have now used PTPROt substrate trapping mutants to identify SYK as a major substrate of this tissue-specific and developmentally regulated PTP. Co-immunoprecipitation studies confirmed that SYK is also an in vivo PTPROt substrate. To examine the effects of PTPROt on SYK-mediated BCR signaling, we generated B-lymphoma cell lines that could be induced to express either wild type PTPROt (PTPROt-wt) or mutant forms of the phosphatase (D/A, C/S) following doxycycline treatment. Dox-induced overexpression of PTPROt-wt inhibited baseline and BCR-triggered SYK phosphorylation whereas the inactive PTPROt mutants had no effect. More specifically, PTPROt-wt overexpression inhibited the phosphorylation of SYK TYR352, the initial TYR residue phosphorylated upon BCR signaling. Thereafter, we showed that overexpression of PTPROt-wt inhibited BCR-induced activation of two direct SYK targets, the SHC and BLNK adaptor proteins, and downstream signaling events including calcium mobilization and MAPK/ERK activation. BCRs also transmit low-level survival signals in the absence of receptor engagement and BCR-proximal PTPs and SYK likely modulate this “tonic” BCR signaling. These observations and the basal phosphorylation of SYK TYR352 in certain B-lymphoma cell lines prompted us to examine the effects of PTPROt-wt and the PTPROt D/A and C/S mutants on B-cell lymphoma proliferation and apoptosis. PTPROt-wt overexpression, but not that of PTPROt D/A or C/S, dramatically inhibited lymphoma cell proliferation and induced apoptosis in the absence of BCR crosslinking. Taken together, these data indicate that PTPROt modulates SYK phosphorylation and tonic BCR signaling and highlights the importance of this pathway for tumor cell survival.

Disclosure: No relevant conflicts of interest to declare.

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