Overexpression of RII beta regulatory subunit of protein kinase A in human colon carcinoma cell induces growth arrest and phenotypic changes that are abolished by site-directed mutation of RII beta.
LS-174T human colon carcinoma cells that contain approximately equal amounts of cAMP-dependent protein kinase (PKA) isozymes, PKA-I and PKA-II, were infected with retroviral vectors coding for regulatory (R) and catalytic (C) subunits of human PKA. In cells overexpressing RII alpha, RII beta and RII beta-P (a RII beta mutant at the autophosphorylation site), PKA-II levels increased while PK-A levels decreased. PKA-I was almost completely eliminated in cells overexpressing RII beta or RII beta-P. In contrast, overexpression of either RI alpha or C alpha had little or no effect on PKA isozyme levels. Although all infectants expressed high levels of PKA subunit mRNAs in accordance with gene introduction, the R subunit protein expression was reflected in PKA isozyme levels rather than in subunit mRNA levels. Only RII beta infectants demonstrated marked growth inhibition in monolayer culture, reduced thymidine incorporation into DNA, and inability to grow in semisolid medium or in serum-free medium. Conversely, all other infectants displayed growth properties similar to uninfected parental cells. The growth-retardation properties of RII beta infectants were reflected in their altered phenotypic appearances. Our findings that the mutant RII beta-P could not mimic the growth-inhibitory effect of RII beta suggest the functional importance of the authophosphorylation site in RII beta. Our results suggest a role for RII beta in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may be involved in neoplastic transformation.
Nesterova
M
M
Celluar Biochemistry Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Yokozaki
H
H
McDuffie
E
E
Cho-Chung
Y S
YS
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D016895:Culture Media, Serum-Free; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D010641:Phenotype; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; C015482:protein kinase modulator; D013936:Thymidine