The tumor suppressor protein P27 blocks cell proliferation by inhibiting cyclin dependent kinases during G1 phase of the cell cycle. Understanding control of p27 tumor suppressing function is essential because its disruption has been linked to aggressive cancers. The p27 protein is heat stable and lacks significant tertiary structure, leading us to hypothesize that enzymatic modification of p27 might alter its association with known binding partners. Transglutaminases are a well known class of enzymes that typically cross link proteins to form barriers. We discovered that bacterial transglutaminase modifies p27 via formation of an intramolecular isopeptide bond between lysine and glutamine. The goal of this work was to determine if human transglutaminase modifies p27 is a similar fashion. A mammalian expression plasmid containing human transglutaminase 2 (hTG2) was obtained and transiently transfected into human h293 cells. Cell extracts were then prepared and analyzed by western blot to confirm hTG2 overexpression. Extracts containing hTG2 were then incubated with purified human p27 to evaluate formation of a novel intramolecular isopeptide bond by looking for a mobility shift on polyacrylamide gels. In addition, hTG2 and p27 were co-transfected into h293 cells to determine if hTG2 can introduce a new isopeptide bond into p27 within cells. These experiments are the first step in determining if tranglutaminase modification of p27 represents a novel regulatory pathway that may influence p27 tumor suppressing function in aggressive human cancers.

Modification of P27 by human transglutaminase

251st National Meeting (2016)

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