Abstract
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 alpha. Using this antibody, the intracellular distribution of endogenous PKI alpha was assessed by immunostaining. The PKI alpha expression and intracellular distribution varied as a function of cell cycle progression. PKI alpha expression appeared low in serum-starved cells and in cells in G1 and increased as cells progressed through S phase. Its distribution became increasingly nuclear as cells entered G2/M. Nuclear levels of PKI alpha remained high through cell division and decreased again as cells reentered G1. The cell cycle regulated expression and nuclear distribution suggests a specific role for PKI alpha in the nucleus during the G2/M phases of the cell cycle. Consistent with this, microinjection of PKI alpha antibody into serum-starved cells prevented their subsequent cell cycle progression. Similarly, overexpression of C subunit in cells arrested at the G1/S boundary prevented their subsequent division. Together these results support the idea that PKI alpha 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.
Highlights
The heat-stable protein kinase inhibitor (PKJ)l is a highly specific inhibitor of the catalytic (C) subunit of cAMP-dependent protein kinase
A polyclonal antibody was raised to purified recombinant PKia and purified on a PKia affinity column
Both serum and the affinity-purified PKia antibody recognized as little as 10 ng of purified recombinant PKia on Western blots (Fig. 1, lane 1)
Summary
The heat-stable protein kinase inhibitor (PKJ)l is a highly specific inhibitor of the catalytic (C) subunit of cAMP-dependent protein kinase. PKI binds to the active site of C with high affinity in the presence of cAMP when Cis dissociated from the holoenzyme complex. Analysis of peptide analogs determined that high affinity binding of PKI to Csubunit could be attributed primarily to 20 residues near the amino terminus. This region contains a cluster of arginine. Residues that mimics the basic subsite present in peptide substrates and a second hydrophobic site required for high affinity binding of PKI to the C subunit. Recent studies demonstrated that PKI entered the nucleus following its injection into the cytoplasm in REF52 cells [12], suggesting a specific role for PKI in the regulation of nuclear stores of C subunit
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