We found that multiple putatively negative regulatory sites were rapidly dephosphorylated as the PTPN6 phosphatase was phosphorylated at an activating site. Inclusion of a mechanism causally linking these events allowed our model to reproduce measured time course data and to generate testable predictions. These predictions were validated experimentally, giving credence to the hypothesized link between PTPN6 activation and dephosphorylation of putatively inhibitory pTyr sites. Our model predicted that loss of PTPN6 would result in sustained phosphorylation of these pTyr sites, and reduction of phosphorylation at other, activating sites. These predictions were confirmed through RNAimediated knockdown of PTPN6 expression and immunoblot measurements with phosphosite-specific antibodies. These results provide strong motivation for future studies of the possible early positive role of PTPN6, ideally in primary cells. We note that a positive role for PTPN6 has been suggested by the results of earlier studies. For example, in vitro, PTPN6 has previously been found to be capable of dephosphorylating the inhibitory C-terminal tyrosine of LCK when the SH2 domain is deleted. The view of PTPN6 as an overall negative regulator of TCR signaling has been based mostly on studies of the motheaten mouse, which is deficient in Ptpn6 and suffers from severe autoimmunity. Recent work has hinted at a more nuanced role. Studies of mice with a T-cell specific Ptpn6 deletion indicate that loss of Ptpn6 in T cells does not lead to overt autoimmunity, nor does it affect the number of memory precursor cells. It has also been found that mechanisms controlling PTPN6 expression are distinct from those controlling other negative regulators of TCR signaling : levels of PTPN6 mRNA and protein are not affected by the miR-181a microRNA, which negatively regulates expression of multiple phosphatases linked to suppression of TCR signaling. Thus, PTPN6 appears to be somewhat enigmatic. Contributing to uncertainty about the function of PTPN6 is an incomplete catalog of its substrates, which is incomplete partly because known substrates do not match an obvious consensus sequence. Our findings, together with those Mepiroxol mentioned above, point to a need to identify signaling proteins whose phosphorylation states are regulated by PTPN6, and to characterize the function of this phosphatase in TCR signaling under precisely controlled conditions. The results presented here suggest that, in Jurkat T cells, PTPN6 has an early positive effect that accelerates signaling, before its negative effects become dominant. The negative effects of PTPN6, such as dephosphorylation of the LCK activation loop, may serve to prevent deleterious overshoots that would otherwise be caused by its positive effects, in addition to setting the baseline level of TCR signaling. As a participant in positive feedback loops, which can act as amplifiers, PTPN6 may also contribute to regulation of Tcell sensitivity. Such a role has been suggested in earlier studies of PTPN6. Several caveats are worth noting. Firstly, although we have demonstrated that PTPN6 acts directly on LCK Y192 and Y505 in vitro, we have not conclusively determined if PTPN6 directly acts on the sites that are observed to undergo dephosphorylation in our proteomics 
experiments, or if instead PTPN6 influences some or all of these sites in an indirect Nodakenin manner. Nonetheless, our knockdown results indicate that PTPN6 positively influences specific events in early signaling, and evidence for a more indirect mechanism would not alter this finding.