A role for the receptor protein tyrosine phosphatase α (PTPα) in immune cell function and regulation of Src family kinases was investigated using thymocytes from PTPα-deficient mice. PTPα-null thymocytes develop normally, but unstimulated PTPα−/− cells exhibit increased tyrosine phosphorylation of specific proteins, increased Fyn activity, and hyperphosphorylation of Cbp/PAG that promotes its association with C-terminal Src kinase. Elevated Fyn activity in the absence of PTPα is due to enhanced phosphorylation of Fyn tyrosines 528 and 417. Some PTPα is localized in lipid rafts of thymocytes, and raft-associated Fyn is specifically activated in PTPα−/− cells. PTPα is not a Cbp/PAG phosphatase, because it is not required for Cbp/PAG dephosphorylation in unstimulated or anti-CD3-stimulated thymocytes. Together, our results indicate that PTPα, likely located in lipid rafts, regulates the activity of raft Fyn. In the absence of PTPα this population of Fyn is activated and phosphorylates Cbp/PAG to enhance association with C-terminal Src kinase. Although TCR-mediated tyrosine phosphorylation was apparently unaffected by the absence of PTPα, the long-term proliferative response of PTPα−/− thymocytes was reduced. These findings indicate that PTPα is a component of the complex Src family tyrosine kinase regulatory network in thymocytes and is required to suppress Fyn activity in unstimulated cells in a manner that is not compensated for by the major T cell PTP and SFK regulator, CD45.

Mice lacking protein tyrosine phosphatase alpha (PTPa) exhibited defects in NMDA receptor (NMDAR)-associated processes such as learning and memory, hippocampal neuron migration, and CA1 hippocampal long-term potentiation (LTP). In vivo molecular effectors linking PTPa and the NMDAR have not been reported. Thus the involvement of PTPa as an upstream regulator of NMDAR tyrosine phosphorylation was investigated in synaptosomes of wild-type and PTPa-null mice. Tyrosine phosphorylation of the NMDAR NR2A and NR2B subunits was reduced upon PTPa ablation, indicating a positive effect of this phosphatase on NMDAR phosphorylation via intermediate molecules. The NMDAR is a substrate of src family tyrosine kinases, and reduced activity of src, fyn, yes and lck, but not lyn, was apparent in the absence of PTPa. In addition, autophosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase linked to NMDAR signaling, was also reduced in PTPa-deficient synaptosomes. Altered protein tyrosine phosphorylation was not accompanied by altered expression of the NMDAR or the above tyrosine kinases at any stage of PTPa-null mouse development examined. In a human embryonic kidney (HEK) 293 cell expression system, PTPa enhanced fyn-mediated NR2A and NR2B tyrosine phosphorylation by several-fold. Together, these findings provide evidence that aberrant NMDAR-associated functions in PTPa-null mice are due to impaired NMDAR tyrosine phosphorylation resulting from the reduced activity of probably more than one of the src family kinases src, fyn, yes and lck. Defective NMDAR activity in these mice may also be linked to the loss of PTPa as an upstream regulator of Pyk2.

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BackgroundThe Transforming Growth Factor-β (TGF-β) regulates myriad cellular events by signaling through members of the Smad family signal transducers. As a key signal transducer of TGF-β, Smad3 exhibits the property of receptor-activated transcriptional modulator and also the novel ability of regulating the proteasomal degradation of two Smad3 interacting proteins, SnoN and HEF1. It has been shown that Smad3 recruits two types of Ub E3 ligases, Smurf2 and the Anaphase Promoting Complex (APC), to mediate SnoN ubiquitination, thereby enhancing SnoN degradation. The molecular mechanisms underlying Smad3-regulated HEF1 degradation are not well understood. Furthermore, it is not clear how Smad3 recruits the APC complex.ResultsWe detected physical interaction between Smad3 and an APC component APC10, as well as the interaction between HEF1 and CDH1, which is the substrate-interacting component within APC. Detailed domain mapping studies revealed distinct subdomains within the MH2 domain of Smad3 for binding to APC10 and HEF1 and suggests the formation of a complex of these four proteins (Smad3, HEF1, APC10 and CDH1). In addition, the protein levels of HEF1 are subjected to the regulation of overexpressed APC10 and CDH1.ConclusionsOur data suggests that Smad3 may recruit the APC complex via a direct interaction with the APC subunit APC10 to regulate the ubiquitination and degradation of its interactor HEF1, which is recognized as an ubiquitination substrate by the CDH1 subunit of the APC complex.