C-terminal Src Kinase-homologous Kinase (CHK), a Unique Inhibitor Inactivating Multiple Active Conformations of Src Family Tyrosine Kinases

Yuh-Ping Chong,Andrew S Chan,Khai-Chew Chan,Nicholas A Williamson,Edwina C Lerner,Thomas E Smithgall,Jeffrey D Bjorge,Donald J Fujita,Anthony W Purcell,Glen Scholz,Terrence D Mulhern,Heung-Chin Cheng

doi:10.1074/jbc.m602951200

Abstract

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.

Highlights

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are two principal endogenous negative regulators of SFKs that inactivate SFKs by phosphorylating their YT
We demonstrate that the CSK-homologous kinase (CHK) is a unique inhibitor capable of inhibiting these and other active conformations of SFKs regardless of their YT phosphorylation status
There are two important outstanding questions arising from our previous findings. (i) Can CHK employ this non-catalytic mechanism to inhibit all active conformations of SFKs? (ii) Can CHK bind to the inactive conformation of SFKs?

Summary

EXPERIMENTAL PROCEDURES

Materials—Wild type CHK expressed in Spodoptera frugiperda 9 (Sf9) cells was purified as described previously [20]. Hck-(222–503) in the column fractions was monitored by the protein kinase activity assay as well as antiFLAG Western blotting. The unbound fractions, containing solely or predominantly pYT-Hck and pYT-Hck(K267M), were analyzed by anti-pYA and anti-pYT Western blotting and mass spectrometry to assess phosphorylation states of these proteins. CHK kinase domain in the column fractions was monitored by Western blotting and kinase activity assay as described previously [20]. To study the ability of CHK to inhibit the pYEEI-activated pYT-Hck, CSK-phosphorylated Hck preparation (1 ␮M) was activated with pYEEI peptide (500 ␮M), under identical conditions described for the co-immunoprecipitation assay. Assay for the Hck(2PA-YEEI) Activity in Lysate of the Transfected HEK293T Cells—HEK293T cells were transiently transfected with the following plasmids: (i) pcDNA3Hck(2PA-YEEI) only, (ii) pcDNA3-CHK and pcDNA3Hck(2PA-YEEI), (iii) pcDNA3-CHK, and (iv) pcDNA3 only, as previously described [20]. Fractions containing radioactivity were analyzed by direct infusion into an Agilent 1100 MSD SL ion trap mass spectrometer fitted with a microionspray source

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS