Earlier studies established that the ability of Csk to inhibit Src-related PTKs in vivo requires intact Csk SH3 and SH2 domains (10
). We subsequently reported that the SH3 domain of Csk mediates highly specific and constitutive interactions with the nonreceptor PTPs PEP and PTP-PEST (11
). In the case of Csk-PEP, this association was shown to augment the capacity of Csk to inactivate Src kinases, by way of the ability of associated PEP to dephosphorylate the activating tyrosine and, possibly, substrates of Src-related enzymes (12
). Several groups also documented that the SH2 domain of Csk can interact with various tyrosine phosphorylated proteins, including Dok-related molecules, PAG, paxillin, and tensin (5
). While the functions of these associations have not been firmly established, they likely mediate the recruitment of Csk to foci of Src-related kinase activation in the cell.
Herein, we found that Csk also associates with PTP-HSCF, the third known member of the PEP family (3
). This interaction was documented in yeast, in a heterologous mammalian cell system, and in primitive hemopoietic cells. Unlike the previously described Csk-PEP and Csk-PTP-PEST interactions, the association between Csk and PTP-HSCF involved the Csk SH2 domain and conserved tyrosines (Y354 and Y381) in the carboxy-terminal noncatalytic region of PTP-HSCF. Transfection studies revealed that Csk and PTP-HSCF cooperated to inhibit signaling events initiated by Src-related PTKs, and that this synergy was greatly facilitated by the domains mediating their association. Finally, evidence was adduced that the inhibitory impact of PTP-HSCF was due at least in part to its capacity to prevent phosphorylation of the positive regulatory tyrosine of Src family kinases.
These findings led to the identification of a novel function for the Csk SH2 domain, distinct from its aforementioned role in recruiting Csk to sites of Src-related PTK activation. By binding the PTP-HSCF, the SH2 region enhanced the capacity of Csk to inhibit Src-related PTKs. This synergism was seemingly consequent to the ability of PTP-HSCF to dephosphorylate the positive regulatory site of Src kinases, thereby ideally complementing the capacity of Csk to phosphorylate their inhibitory carboxy-terminal tyrosine. Such a mechanism does not exclude the possibility that PTP-HSCF also served the purpose of recruiting Csk near activated Src-related molecules. In fact, this is likely to be the case, as tyrosine phosphorylation of PTP-HSCF is triggered by activated Src kinases (this report) (38
). Hence, binding of Csk to tyrosine phosphorylated PTP-HSCF probably fulfils two complementary goals: juxtaposition of Csk near activated Src kinases by physical recruitment and enhancement of the inhibitory potential of Csk by a catalytic mechanism.
Obviously, the interaction of Csk with PTP-HSCF is evocative of its association with PEP and PTP-PEST. One important difference, however, is that the binding to PEP and PTP-PEST is mediated by the SH3 domain of Csk and is constitutive (11
). By opposition, the association of Csk with PTP-HSCF was found to be SH2 domain mediated and reversible. It could be implied from this distinction that the inhibitory signal transduced by Csk-PTP-HSCF is qualitatively different from that triggered by Csk-PEP and Csk-PTP-PEST. Whereas this notion remains plausible, our studies failed to produce any evidence supporting this notion. Rather, the SH2 domain- and SH3 domain-dependent associations of Csk with PTPs seem to result in functionally analogous inhibitory mechanisms. The capacity of PTP-HSCF (this report) and PEP (12
) to inactivate Src kinases through a similar biochemical effect is in keeping with this proposition. The phosphotyrosine-dependence of the Csk-PTP-HSCF association may simply indicate that it is of shorter duration, as the complex would dissociate once the activity of the Src kinases is repressed and tyrosine phosphorylation of PTP-HSCF has subsided. Consequently, inhibition by Csk-PTP-HSCF may be more finely tuned to the degree of activation of Src family PTKs.
Our data revealed that the presence of Csk also favored the tyrosine phosphorylation of PTP-HSCF. Interestingly, this effect was found to be independent of the catalytic activity of Csk. Rather, it required the presence of an intact Csk SH2 domain. Binding of the Csk SH2 domain probably stabilized tyrosine phosphorylation of PTP-HSCF, by shielding the phosphotyrosines from dephosphorylation by PTP-HSCF and/or other cellular PTPs. A similar situation has been documented for other SH2 domains (20
). Through this mechanism, the association of Csk with PTP-HSCF would trigger an amplifying loop that augments Csk recruitment and allows a more sustained inhibitory response to take place. It is likely that this feature is a common and advantageous consequence of SH2 domain-mediated interactions.
Since tyrosine phosphorylation of PTP-HSCF was difficult to detect in the absence of PTP inhibition, one could argue that such a phosphorylation does not occur in a physiologically meaningful way. However, several findings indicated that this is unlikely to be the case. First, tyrosine phosphorylation of PTP-HSCF was highly specific, as we were unable to detect a similar modification of PEP or PTP-PEST (our unpublished results). Second, tyrosine phosphorylation of PTP-HSCF could be detected in the absence of PTP inhibition, such as when the levels of Csk were increased in the cell (Fig. ). Importantly, this phosphorylation occurred at the sites that were also phosphorylated under conditions of PTP inhibition (our unpublished results). And third, the putative sites of tyrosine phosphorylation of PTP-HSCF were essential for the ability of wild-type PTP-HSCF and Csk to cooperate towards inhibiting Src-related PTKs. Thus, tyrosine phosphorylation of PTP-HSCF is very likely to occur in cells, albeit transiently, and to be a biologically significant event.
The results of our studies allowed the identification of two potential binding motifs for the Csk SH2 domain: Y354
AVV and Y381
SQV of PTP-HSCF. It was previously demonstrated that the sequence Y314
SSV in PAG was also recognized by the SH2 domain of Csk (5
). These three sequences are in clear agreement with the motif pY(T/A/S)X(M/I/V) (where pY is phosphotyrosine and X is any residue), which has been selected as the preferential Csk SH2 domain-binding sequence in a peptide library (37
). Hence, it is probable that the peptide library-derived motif represents a predominant Csk SH2 domain-binding sequence in vivo. Obviously, it will be of interest to see whether other Csk SH2 region-binding proteins, such as Dok-related molecules, paxillin, tensin, and Fak, associate with Csk through a similar decoy.
Chk is a Csk-related molecule selectively expressed in hemopoietic cells and brain (9
). Interestingly, we found herein that Chk did not bind to PTP-HSCF. Likewise, it was reported earlier that Chk was incapable of associating with PEP and PTP-PEST (11
). Therefore, the ability to interact physically with the PEP family of PTPs appears to be restricted to Csk. Even though the biological significance of this distinction remains to be fully elucidated, it is noteworthy that Csk and Chk do seem to have dissimilar biological roles. In support of this idea, it was found that Chk was an inefficient negative regulator of antigen receptor signaling in T-cells, in striking contrast to Csk (15
). The inability of Chk to interact with PEP-related phosphatases may explain at least part of this functional difference.
In combination with previously published findings (11
), the results reported here reveal that Csk associates with all three members of the PEP family. This is probably more than a coincidence. As there is no conclusive evidence that Csk interacts with other PTPs (our unpublished results), this observation strongly argues for a unique functional affinity between the two classes of molecules. Very possibly, their alliance has evolved from their shared capacity to inhibit Src-related PTKs. As a corollary, the selective nature of the association of Csk with PEP family members suggests that PEP-related PTPs play a significant role in the regulation of Src-related PTKs in vivo. Along these lines, it should be mentioned that, with the exception of the receptor-like PTP CD45 and members of the PEP family (12
; this report), little is known of the PTPs responsible for inactivating Src kinases in mammalian cells. In light of our data, it seems probable that PEP-related molecules play a pivotal role in this process. If this is the case, it will be interesting to determine whether the three distinct types of Csk-PTP complexes have independent functions. Our studies to date suggest that the Csk-PEP and Csk-PTP-HSCF complexes are capable of similar inhibitory effects on Src kinases. However, taking into consideration the reported differences in the intracellular localization of PEP, PTP-PEST, and, possibly, PTP-HSCF (11
), these three complexes may be aimed at inhibiting separate cellular pools of Src-related kinases. Future studies will be needed to address this possibility.