A novel non-catalytic mechanism employed by the C-terminal Src-homologous kinase to inhibit Src-family kinase activity.
Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (Tyr(T)), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating Tyr(T), CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic Tyr(T)-independent mechanism because it occurs even in the absence of ATP or when Tyr(T) of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK.Src complex exists in rat brain, and recombinant CHK.Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their Tyr(T) and (ii) by this novel Tyr(T)-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their Tyr(T) is phosphorylated. Thus, the ability of the Tyr(T)-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances.
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
Yuh-Ping
YP
Chong
Terrence D
TD
Mulhern
Hong-Jian
HJ
Zhu
Donald J
DJ
Fujita
Jeffrey D
JD
Bjorge
John-Paul
JP
Tantiongco
Nikolaos
N
Sotirellis
Daisy Sio Seng
DS
Lio
Glen
G
Scholz
Heung-Chin
HC
Cheng
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009419:Nerve Tissue Proteins; D010281:Parathyroid Hormone; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry* / Q000378:metabolism*; D015183:Restriction Mapping; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
C473930:ARHGEF26 protein, human; D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D010281:Parathyroid Hormone; D010446:Peptide Fragments; C023443:carboxyl-terminal parathyroid hormone; C091243:Matk protein, rat; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases