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. Ser(114) (the autophosphorylation site) of human RII(beta) was replaced with Ala (RII(beta)-P) or Arg(264) 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 Rnp. 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).