Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RIIβ regulatory subunit to lose its ability to revert transformed fibroblasts

Alfredo Budillon, Anna Cereseto, Andrey Kondrashin, Maria Nesterova, Giorgio Merlo, Timothy Clair, Yoon S. Cho-Chung

Research output: Contribution to journalArticle

Abstract

The RIIβ 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 RIIβ by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RIIβ was replaced with Ala (RIIβ-P) or Arg264 of KKRK was replaced with Met (RIIβ-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RIIβ, RIIβ-P, and RIIβ-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. Overexpression of wild-type or mutant RIIβ resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RIIβ cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RIIβ overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RIIβ. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RIIβ-P or RIIβ-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 RIIβ.

Original languageEnglish
Pages (from-to)10634-10638
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number23
Publication statusPublished - Nov 7 1995

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Cyclic AMP-Dependent Protein Kinases
Point Mutation
Protein Kinases
Fibroblasts
Growth
Isoenzymes
NIH 3T3 Cells
Protein Sorting Signals
Site-Directed Mutagenesis
8-chloro-cyclic adenosine monophosphate

ASJC Scopus subject areas

  • General
  • Genetics

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Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RIIβ regulatory subunit to lose its ability to revert transformed fibroblasts. / Budillon, Alfredo; Cereseto, Anna; Kondrashin, Andrey; Nesterova, Maria; Merlo, Giorgio; Clair, Timothy; Cho-Chung, Yoon S.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 92, No. 23, 07.11.1995, p. 10634-10638.

Research output: Contribution to journalArticle

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