Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts.
Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.
Laboratoire de Physiopathologie du Développement, Paris, France.
S
S
Tournier
F
F
Raynaud
P
P
Gerbaud
S M
SM
Lohmann
M
M
Dorée
D
D
Evain-Brion
D000328:Adult; D000595:Amino Acid Sequence; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts; D006801:Humans; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*
D011494:Protein Kinases; D016203:CDC2 Protein Kinase