SH2 Domain-Mediated Interaction of Inhibitory Protein Tyrosine Kinase Csk with Protein Tyrosine Phosphatase-HSCF

Abstract

The protein tyrosine kinase (PTK) Csk is a potent negative regulator of several signal transduction processes, as a consequence of its exquisite ability to inactivate Src-related PTKs. This function requires not only the kinase domain of Csk, but also its Src homology 3 (SH3) and SH2 regions. We showed previously that the Csk SH3 domain mediates highly specific associations with two members of the PEP family of nonreceptor protein tyrosine phosphatases (PTPs), PEP and PTP-PEST. In comparison, the Csk SH2 domain interacts with several tyrosine phosphorylated molecules, presumed to allow targetting of Csk to sites of Src family kinase activation. Herein, we attempted to understand better the regulation of Csk by identifying ligands for its SH2 domain. Using a modified yeast two-hybrid screen, we uncovered the fact that Csk associates with PTP-HSCF, the third member of the PEP family of PTPs. This association was documented not only in yeast cells but also in a heterologous mammalian cell system and in cytokine-dependent hemopoietic cells. Surprisingly, the Csk-PTP-HSCF interaction was found to be mediated by the Csk SH2 domain and two putative sites of tyrosine phosphorylation in the noncatalytic portion of PTP-HSCF. Transfection experiments indicated that Csk and PTP-HSCF synergized to inhibit signal transduction by Src family kinases and that this cooperativity was dependent on the domains mediating their association. Finally, we obtained evidence that PTP-HSCF inactivated Src-related PTKs by selectively dephosphorylating the positive regulatory tyrosine in their kinase domain. Taken together, these results demonstrate that part of the function of the Csk SH2 domain is to mediate an inducible association with a PTP, thereby engineering a more efficient inhibitory mechanism for Src-related PTKs. Coupled with previously published observations, these data also establish that Csk forms complexes with all three known members of the PEP family.

PSTPIP2, a Protein Associated with Autoinflammatory Disease, Interacts with Inhibitory Enzymes SHIP1 and Csk

“…The most likely candidate for bringing this additional Csk to the complex is PTP-PEP, which interacted with PSTPIP2 in an inducible manner. However, another option is that PTP-HSCF, which, unlike PTP-PEST or PTP-PEP, binds Csk via phosphorylated tyrosines in its C-terminal tail (22), is responsible for this inducible binding.…”

“…20). All three family members bind Csk via proline-rich or tyrosine-containing motifs in their central region (18,21,22). In addition, a conserved C-terminal homology domain of PEST-PTPs binds two related proteins, PSTPIP1 and PSTPIP2 (23,24).…”

Cellular Functions of Mitogen-activated Protein Kinases and Protein Tyrosine Phosphatases in Ovarian Granulosa Cells

“…In addition to p190 RhoGAP, Pro, Ser, Thr phosphatase interacting protein 1 (PSTPIP1) and the cytoplasmic PTKs, c-Abl and the Src-related kinases, are known substrates for PTP20 [39][40][41]. PSTPIP1 interacts with and serves as a substrate for c-Abl, and can bridge interactions between c-Abl and PTP20 with the dephosphorylation of c-Abl by PTP20 [40].…”

Uncleaved BAP31 in Association with A4 Protein at the Endoplasmic Reticulum Is an Inhibitor of Fas-initiated Release of Cytochromec from Mitochondria

“…Identification of Clones-All yeast transformations were done as described (31). Routinely, 20 g of DNA of each library was transformed into pRSB-L40 and clones were selected on leucine/tryptophane/histidine plates in the presence of 0.2 mM CuSO 4 and 5 mM 3-aminotriazole.…”