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 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.
Department of Chemistry, University of California at San Diego, La Jolla 92093.
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Wen
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Taylor
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Meinkoth
D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology*; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008938:Mitosis; D011994:Recombinant Proteins
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C015482:protein kinase modulator