Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.
The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.
Lilly
L
Wong
Scot A
SA
Lieser
Osamu
O
Miyashita
Meghan
M
Miller
Kjetil
K
Tasken
Josè N
JN
Onuchic
Joseph A
JA
Adams
Virgil L
VL
Woods
Patricia A
PA
Jennings
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002384:Catalysis; D002645:Chickens; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D009038:Motion; D010446:Peptide Fragments; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology*; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; C498710:PAG1 protein, human; D010446:Peptide Fragments; D010750:Phosphoproteins; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human