P27 is a tumor-suppressor protein involved in cell cycle regulation, functioning by binding to and inhibiting cyclin-dependent kinases (CDKs). Surprisingly, the p27 gene is seldom mutated or deleted; instead, p27 is deregulated at the protein level. This, in turn, represses the function of the p27 protein, and thus contributes to the development of several types of aggressive cancers. It is due to this that understanding the mechanisms surrounding the function of the p27 protein is essential. The p27 protein is heat stable and lacks a tertiary structure, leaving a structural change as the most likely method of suppressing its function. Thus, we hypothesized that imparting structure to the polypeptide resulted in the compromise of function. In searching for an enzyme that had the ability to alter protein structure, we identified transglutaminase, which catalyzes protein cross-linking via the formation of an isopeptide bond between lysine and glutamine groups. Additionally, altered levels of both nuclear and cellular transglutaminase have been observed in cancer cells. Knowing this, we incubated purified p27 and transglutaminase, then analyzed p27 mobility on a denaturing polyacrylamide gel. Interestingly, p27 monomers were not crosslinked. Instead, p27 migrated faster, suggesting a conformational change due to intramolecular isopeptide bond formation. The kinetics, inhibition, and specificity of this novel reaction have been evaluated. Additionally, we have analyzed p27 with select binding partners, such as GRB2 and cyclin E. Our results identify a possible link between transglutaminase modification of p27 and proliferation of aggressive cancers.

Modification of p27kip1 by transglutaminase

251st National Meeting (2016)

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