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Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A.

Zwaenepoel K, Louis JV, Goris J, Janssens V - BMC Genomics (2008)

Bottom Line: The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue.We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Protein Phosphorylation and Proteomics Group, Dept, Molecular Cell Biology, Faculty of Medicine, K,U, Leuven, Gasthuisberg O&N1, Herestraat 49 box 901, B-3000 Leuven, Belgium. Karen.Zwaenepoel@med.kuleuven.be

ABSTRACT

Background: Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised.

Results: We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.

Conclusion: In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.

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Diversity of the murine PR130/72 and PR59 isoforms. A: Autoradiography of [35S]-labelled in vitro transcription-translation products of plasmids (1 μg) containing cDNA of murine PR59/B"δ1 (lane δ1), PR59/B"δ2 (lane δ2) and PR59/B"δ3 (lane δ3). B: Immunoprecipitation of mPR72/PR130 isoforms from different murine cells and tissues (N = NIH 3T3, H = heart, B = brain, A = adrenal gland) using pre-immunisation sera (as negative control) and three different PR72/PR130 antibodies: PR72rec. AB recognizes all putative PR72 isoforms and the PR130 isoforms which contain the common C-terminal region, PR130N rec. AB recognizes all PR130 isoforms, and PR72N pept. AB recognizes all PR72 isoforms.
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Figure 1: Diversity of the murine PR130/72 and PR59 isoforms. A: Autoradiography of [35S]-labelled in vitro transcription-translation products of plasmids (1 μg) containing cDNA of murine PR59/B"δ1 (lane δ1), PR59/B"δ2 (lane δ2) and PR59/B"δ3 (lane δ3). B: Immunoprecipitation of mPR72/PR130 isoforms from different murine cells and tissues (N = NIH 3T3, H = heart, B = brain, A = adrenal gland) using pre-immunisation sera (as negative control) and three different PR72/PR130 antibodies: PR72rec. AB recognizes all putative PR72 isoforms and the PR130 isoforms which contain the common C-terminal region, PR130N rec. AB recognizes all PR130 isoforms, and PR72N pept. AB recognizes all PR72 isoforms.

Mentions: Since mPR59/B"δ1 and mPR59/B"δ3 are putatively novel, and to further confirm their existence, we performed a reverse transcription on NIH 3T3 and murine heart RNA using primer E3 annealing in the mPR59 3' UTR. With the resulting cDNAs as templates and the use of five different primers, annealing with the specific N-termini of PR59/B"δ1–4 (Bδ1, Bδ2, Bδ3/4) and/or the specific C-termini of PR59/B"δ1–3 (E2) and PR59/B"δ4 (E1), we were able to generate several DNA fragments, which after sequencing, were shown to correspond to PR59/B"δ1, PR59/B"δ2 and PR59/B"δ3. In vitro transcription-translation reactions of these cDNAs revealed proteins of about 60 kDa (PR59/B"δ1), 55 kDa (PR59/B"δ2) and 40 kDa (PR59/B"δ3) (Figure 1A), implying that not only the messengers of PR59/B"δ1,δ2,δ3 are present in murine cells, but that they can also be translated into proteins. A cDNA for mPR59/B"δ4 could not be amplified using this approach, confirming its low occurrence in the EST database.


Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A.

Zwaenepoel K, Louis JV, Goris J, Janssens V - BMC Genomics (2008)

Diversity of the murine PR130/72 and PR59 isoforms. A: Autoradiography of [35S]-labelled in vitro transcription-translation products of plasmids (1 μg) containing cDNA of murine PR59/B"δ1 (lane δ1), PR59/B"δ2 (lane δ2) and PR59/B"δ3 (lane δ3). B: Immunoprecipitation of mPR72/PR130 isoforms from different murine cells and tissues (N = NIH 3T3, H = heart, B = brain, A = adrenal gland) using pre-immunisation sera (as negative control) and three different PR72/PR130 antibodies: PR72rec. AB recognizes all putative PR72 isoforms and the PR130 isoforms which contain the common C-terminal region, PR130N rec. AB recognizes all PR130 isoforms, and PR72N pept. AB recognizes all PR72 isoforms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529318&req=5

Figure 1: Diversity of the murine PR130/72 and PR59 isoforms. A: Autoradiography of [35S]-labelled in vitro transcription-translation products of plasmids (1 μg) containing cDNA of murine PR59/B"δ1 (lane δ1), PR59/B"δ2 (lane δ2) and PR59/B"δ3 (lane δ3). B: Immunoprecipitation of mPR72/PR130 isoforms from different murine cells and tissues (N = NIH 3T3, H = heart, B = brain, A = adrenal gland) using pre-immunisation sera (as negative control) and three different PR72/PR130 antibodies: PR72rec. AB recognizes all putative PR72 isoforms and the PR130 isoforms which contain the common C-terminal region, PR130N rec. AB recognizes all PR130 isoforms, and PR72N pept. AB recognizes all PR72 isoforms.
Mentions: Since mPR59/B"δ1 and mPR59/B"δ3 are putatively novel, and to further confirm their existence, we performed a reverse transcription on NIH 3T3 and murine heart RNA using primer E3 annealing in the mPR59 3' UTR. With the resulting cDNAs as templates and the use of five different primers, annealing with the specific N-termini of PR59/B"δ1–4 (Bδ1, Bδ2, Bδ3/4) and/or the specific C-termini of PR59/B"δ1–3 (E2) and PR59/B"δ4 (E1), we were able to generate several DNA fragments, which after sequencing, were shown to correspond to PR59/B"δ1, PR59/B"δ2 and PR59/B"δ3. In vitro transcription-translation reactions of these cDNAs revealed proteins of about 60 kDa (PR59/B"δ1), 55 kDa (PR59/B"δ2) and 40 kDa (PR59/B"δ3) (Figure 1A), implying that not only the messengers of PR59/B"δ1,δ2,δ3 are present in murine cells, but that they can also be translated into proteins. A cDNA for mPR59/B"δ4 could not be amplified using this approach, confirming its low occurrence in the EST database.

Bottom Line: The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue.We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Protein Phosphorylation and Proteomics Group, Dept, Molecular Cell Biology, Faculty of Medicine, K,U, Leuven, Gasthuisberg O&N1, Herestraat 49 box 901, B-3000 Leuven, Belgium. Karen.Zwaenepoel@med.kuleuven.be

ABSTRACT

Background: Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised.

Results: We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.

Conclusion: In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.

Show MeSH
Related in: MedlinePlus