The heat-stable protein kinase inhibitor (PKI) is a potent and specific inhibitor of the catalytic (C) subunit of the cAMP-dependent protein kinase. We report the isolation of a polyclonal antibody raised to purified recombinant PKI. Using this antibody, the intracellular distribution of endogenous PKI was assessed by immunostaining. The PKI expression and intracellular distribution varied as a function of cell cycle progression. PKI expression appeared low in serum-starved cells and in cells in G and increased as cells progressed through S phase. Its distribution became increasingly nuclear as cells entered G/M. Nuclear levels of PKI remained high through cell division and decreased again as cells reentered G. The cell cycle regulated expression and nuclear distribution suggests a specific role for PKI in the nucleus during the G/M phases of the cell cycle. Consistent with this, microinjection of PKI antibody into serum-starved cells prevented their subsequent cell cycle progression. Similarly, overexpression of C subunit in cells arrested at the G/S boundary prevented their subsequent division. Together these results support the idea that PKI plays an important role in the inhibition of nuclear C subunit activity required for cell cycle progression, although a determination of the relative amounts of endogenous nuclear PKI and C-subunit will be required to substantiate this hypothesis.

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