Abstract
Differential association of regulatory B subunits with a core heterodimer, composed of a catalytic (C) and a structural (A) subunit, is an important mechanism that regulates protein phosphatase 2A (PP2A). We have isolated and characterized three novel cDNAs related to the B' subunit of bovine cardiac PP2A. Two human (B'alpha1 and B'alpha2) and a mouse (B'alpha3) cDNA encode for alternatively spliced variants of the B subunit. The deduced primary sequences of these clones contain 12 of 15 peptides derived from the purified bovine B' subunit. Differences between the deduced sequences of the B alpha splice variants and the cardiac peptide sequences suggest the existence of multiple isoforms of the B' subunit. Comparison of the protein and nucleotide sequences of the cloned cDNAs show that all three forms of B'alpha diverge at a common splice site near the 3'-end of the coding regions. Northern blot and reverse transcription-polymerase chain reaction analyses revealed that the B'alpha transcripts (4.3-4.4 kb) are widely expressed and very abundant in heart and skeletal muscle. The expressed human and mouse B'alpha proteins readily associated with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Immunofluorescence microscopy revealed that epitope-tagged B'alpha was localized in both the cytosol and nuclei of transiently transfected cells. The efficiency of binding of all three expressed proteins to a glutathione S-transferase-A subunit fusion protein was greatly enhanced by the addition of the C subunit. Expression of the B'alpha subunits in insect Sf9 cells resulted in formation of AC.B'alpha heterotrimers with the endogenous insect A and C subunits. These results show that the B' subunit, which is the predominant regulatory subunit in cardiac PP2A, is a novel protein whose sequence is unrelated to other PP2A regulatory subunits. The nuclear localization of expressed B'alpha suggests that some variants of the B' subunit are involved in the nuclear functions of PP2A.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U37352 and U37353
An 854-bp fragment was amplified by polymerase chain reaction (PCR) as described previously [13] from a human umbilical vein epithelial cell (HUVEC) cDNA library
Molecular Cloning of cDNAs Encoding BЈ␣—The major form of regulatory subunit in purified bovine cardiac phosphatase 2A (PP2A) is related to the BЈ subunit originally identified in a form of rabbit muscle PP2A termed PP2A0 [17]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U37352 and U37353. There are at least five distinct families of proteins that interact with and regulate the PP2A core enzyme These include the B, BЈ, PR72 (BЉ), and ␦ families of regulatory subunits and the small and middle tumor antigens of DNA tumor viruses [8]. Multiple isoforms (␣, , ␥) of the B subunit and a splice variant of B␣ have been isolated from mammalian sources [9, 16, 17] These proteins are 81– 87% identical and diverge primarily at the amino termini. We describe the isolation and expression of novel cDNAs encoding human (BЈ␣1 and BЈ␣2) and mouse (BЈ␣3) members of the BЈ familiy of PP2A regulatory subunits. The BЈ␣ subunits may be important for the localization/translocation of PP2A into the nucleus
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