THE EXPRESSION AND INTRACELLULAR-DISTRIBUTION OF THE HEAT-STABLE PROTEIN-KINASE INHIBITOR IS CELL-CYCLE-REGULATED

The heat stable protein kinase inhibitor (PKI) is a potent and specifi c inhibitor of the catalytic (C) subunit of the cAMP-dependent protein kinase, We report the isolation of a polyclonal antibody raised to pu rified recombinant PKI alpha. Using this antibody, the intracellular d istribution of endogenous PKI alpha was assessed by immunostaining. Th e PKI alpha expression and intracellular distribution varied as a func tion of cell cycle progression. PKI alpha expression appeared low in s erum starved cells and in cells in G(1) and increased as cells progres sed through S phase, Its distribution became increasingly nuclear as c ells entered G(2)/M. Nuclear levels of PKI alpha remained high through cell division and decreased again as cells reentered G(1). The cell c ycle regulated expression and nuclear distribution suggests a specific role for PKI alpha in the nucleus during the G(2)/M phases of the cel l cycle, Consistent with this, microinjection of PKI alpha antibody in to serum-starved cells prevented their subsequent cell cycle progressi on, Similarly, overexpression of C subunit in cells arrested at the G( 1)/S boundary prevented their subsequent division, Together these resu lts support the idea that PKI alpha plays an important role in the inh ibition of nuclear C subunit activity required for cell cycle progress ion, although a determination of the relative amounts of endogenous nu clear PKI and C-subunit will be required to substantiate this hypothes is.