Interaction between the amino-terminal SH3 domain of CRK and its natural target proteins.
CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), Shc, and paxillin. The SH3 domain, in turn, binds to cytosolic proteins of 135-145, 160, 180, and 220 kDa. We screened expression libraries by Far Western blotting, using CRK SH3 as a probe, and identified partial cDNA sequences of four distinct proteins, including C3G, DOCK180, EPS15, and clone ST12. The consensus sequence of the CRK SH3 binding sites as deduced from their amino acid sequences was Pro+3-Pro+2-X+1-Leu0-Pro-1-X-2-Lys-3. The interaction of the CRK SH3 domain with the DOCK180 peptide was examined with an optical biosensor, based on the principles of surface plasmon resonance. A low dissociation constant of the order of 10(-7) resulted from a high association rate constant (kassoc = 3 x 10(4)) and low dissociation rate constant (kdiss = 3 x 10(-3)). All CRK-binding proteins except clone ST12 also bound to another adaptor protein, Grb2. Mutational analysis revealed that glycine at position +1 of ST12 inhibited the binding to Grb2 while retaining the high affinity binding to CRK SH3. The result suggests that the amino acid at position +1 also contributes to the high affinity binding of the peptides to the SH3 domain of Grb2, but not to that of CRK.
Matsuda
M
M
Department of Pathology, National Institute of Health, Toyama, Shinjuku-ku, Tokyo 162, Japan.
Ota
S
S
Tanimura
R
R
Nakamura
H
H
Matuoka
K
K
Takenawa
T
T
Nagashima
K
K
Kurata
T
T
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002645:Chickens; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D015870:Gene Expression; D015723:Gene Library; D005982:Glutathione Transferase / Q000096:biosynthesis; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D050736:Oncogene Protein v-crk; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011392:Proline; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry; D020744:rac GTP-Binding Proteins; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C099818:DOCK1 protein, human; C486206:EPS15 protein, human; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D050736:Oncogene Protein v-crk; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D011392:Proline; D005982:Glutathione Transferase; D020744:rac GTP-Binding Proteins