Related Experiment Video

Updated: May 11, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal

R Jove, S Kornbluth, H Hanafusa

    Cell
    |September 11, 1987
    PubMed

    Insights

    Phosphorylation of cellular src protein (p60c-src) at tyrosine 527 is crucial for regulating its activity. This study shows tyrosine 527 phosphorylation occurs independently of p60c-src kinase activity.

    Area of Science:

    • Molecular Biology
    • Cellular Signaling
    • Oncogenesis

    Background:

    • Cellular src protein (p60c-src) is a non-receptor tyrosine kinase.
    • Phosphorylation at tyrosine 527 (Y527) is known to suppress p60c-src kinase activity and its transforming potential.
    • The dependency of Y527 phosphorylation on p60c-src's own kinase activity remains unclear.

    Purpose of the Study:

    • To investigate whether the phosphorylation of tyrosine 527 in p60c-src is dependent on its intrinsic protein-tyrosine kinase activity.
    • To elucidate the mechanism regulating p60c-src activity and its role in cellular transformation.

    Main Methods:

    • Site-directed mutagenesis was used to create a catalytically inactive mutant of chicken p60c-src by substituting lysine 295 with methionine (p60c-src(M295)).
    • The expression and phosphorylation patterns of both wild-type p60c-src and the p60c-src(M295) mutant were analyzed in chicken cells and yeast.

    Main Results:

    • The p60c-src(M295) mutant exhibited no detectable protein-tyrosine kinase activity in both chicken cells and yeast.
    • Phosphorylation of p60c-src(M295) on tyrosine and serine residues in chicken cells was comparable to that of wild-type p60c-src.
    • However, p60c-src(M295) failed to undergo tyrosine phosphorylation when expressed in yeast.

    Conclusions:

    • Tyrosine 527 phosphorylation of p60c-src is independent of its own kinase activity.
    • A trans-acting protein kinase, present in chicken cells but absent in yeast, is responsible for phosphorylating tyrosine 527.
    • These findings suggest that a kinase distinct from p60c-src mediates Y527 phosphorylation, offering insights into src kinase regulation.

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