Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.
The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D006801:Humans; D007527:Isoenzymes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000235:genetics*
D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D007527:Isoenzymes; C517837:PRKAR2B protein, human; D021382:Protein Sorting Signals; D015124:8-Bromo Cyclic Adenosine Monophosphate; C044258:8-chloro-cyclic adenosine monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases