Csk homologous kinase (CHK) and ErbB-2 interactions are directly coupled with CHK negative growth regulatory function in breast cancer.
Our previous studies demonstrated that Csk homologous kinase (CHK) acts as a negative growth regulator of human breast cancer through inhibition of ErbB-2/neu-mediated Src family kinase activity (Bougeret, C., Jiang, S., Keydar, I., and Avraham, H. (2001) J. Biol. Chem. 276, 33711-33720. The interaction between the CHK SH2 domain and Tyr(P)(1248) of the ErbB-2 receptor has been shown to be specific and critical for CHK function. In this report, we investigated whether the interaction of the CHK SH2 domain and ErbB-2 is directly related to the inhibition of heregulin-stimulated Src kinase activity. We constructed three CHK SH2 domain binding mutants: G129R (enhanced binding), R147K (inhibited binding), and R147A (disrupted binding). NMR spectra for the domains of each construct were used to evaluate their interaction with a Tyr(P)(1248)-containing ErbB-2 peptide. G129R showed enhanced binding to ErbB-2, whereas binding was completely disrupted by R147A. The enhanced binding mutant showed chemical shift changes at the same residues as wild-type CHK, indicating that this mutant has the same binding characteristics as the wild-type protein. Furthermore, inhibition of heregulin-stimulated Src kinase activity was markedly diminished by R147A, whereas G129R-mediated inhibition was stronger as compared with wild-type CHK. These results indicate that the specific interaction of CHK and ErbB-2 via the SH2 domain of CHK is directly related to the growth inhibitory effects of CHK. These new CHK high affinity binding constructs may serve as good candidates for inhibition of the ErbB-2/Src transduction pathway in gene therapy studies in breast cancer.
Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA.
Soyoun
S
Kim
Radoslaw
R
Zagozdzon
Alan
A
Meisler
James D
JD
Baleja
Yigong
Y
Fu
Shalom
S
Avraham
Hava
H
Avraham
D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000201:enzymology*; D002455:Cell Division; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010452:Peptide Biosynthesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019061:src-Family Kinases; C085377:MATK protein, human