Csk-mediated phosphorylation of substrates is regulated by substrate tyrosine phosphorylation.
Csk is a cellular protein tyrosine kinase (PTK) that has been shown to specifically regulate the activity of Src kinase family members by phosphorylation of a carboxy-terminal tyrosine residue. The molecular mechanisms controlling Csk regulation and its substrate specificity have not been elucidated. Here we report a novel type of overlay kinase assay that allows to probe for Csk-mediated phosphorylation of cellular substrates separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Most of the cell lines analyzed with this method revealed only a few potential Csk substrates. However, an increased number of Csk substrates was detected in NIH3T3 cells expressing a constitutively activated form of the Src kinase Lck or in PC12 and NIH3T3 cells that had been treated with pervanadate. These cells all display an increased level of cellular protein tyrosine phosphorylation which led to the conclusion that Csk preferentially phosphorylates tyrosine-phosphorylated proteins. To verify this hypothesis we analyzed Csk-mediated phosphorylation of recombinant Lck, a known Csk substrate. Results demonstrated that autophosphorylation of Lck (at Tyr394) facilitates Csk-mediated phosphorylation of Lck at its regulatory site (Tyr505). Subsequent peptide binding studies revealed that Csk can bind to a peptide corresponding to the Lck-autophosphorylation site only when it is phosphorylated. These findings suggest that autophosphorylation of Lck at Tyr394 triggers an interaction with Csk and thereby facilitates subsequent phosphorylation and inactivation of Lck. The phosphorylation of other cellular Csk substrates may be regulated by a similar mechanism.
Amrein
K E
KE
Department of Metabolic Diseases, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.
Molnos
J
J
zur Hausen
J D
JD
Flint
N
N
Takacs
B
B
Burn
P
P
D016475:3T3 Cells; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases
D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases