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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.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.

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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Association of the main human and murine PR72/B" isoforms with PP2AD. COS7 cells were transfected with GST, hPR130-GST, hPR72-GST, hPR70-GST, mG5PR/B"γ-GST, mPR59/B"δ1-GST, mPR59/B"δ2-GST and mPR59/B"δ3-GST. 48 h after transfection, a GST pull down assay was performed and binding of PR65/A and C subunits was evaluated via Western blotting using specific antibodies.
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Figure 5: Association of the main human and murine PR72/B" isoforms with PP2AD. COS7 cells were transfected with GST, hPR130-GST, hPR72-GST, hPR70-GST, mG5PR/B"γ-GST, mPR59/B"δ1-GST, mPR59/B"δ2-GST and mPR59/B"δ3-GST. 48 h after transfection, a GST pull down assay was performed and binding of PR65/A and C subunits was evaluated via Western blotting using specific antibodies.

Mentions: With the exception of XN73 and mPR130/B"α3, all B" family members contain a conserved region and specific N- and C-termini. The conserved region, which contains two ASBDs and two EF-hand motifs, is necessary for PR65/A binding [3]. Since the first ASBD is not intact in mPR59/B"δ3 (Figure 2), we wondered whether this isoform is still able to bind PP2A. To this end, we made GST-fusion proteins of the main B" family members and overexpressed these in COS-7 cells. After a GST-pull down assay, we evaluated the binding of both the PR65/A and catalytic subunit of PP2A via Western blotting. As expected, all main B" isoforms (hPR130, hPR72, hPR70, mG5PR/B"γ, mPR59/B"δ1 and mPR59/B"δ2) bind PP2A (Figure 5), and are therefore genuine B" subunits. In contrast, mPR59/B"δ3 fails to bind PP2A, suggesting it is not a regulatory subunit of PP2A (Figure 5). Like XN73 and mPR130/B"α3, it might be involved in regulation of PP2A by competing with the other mPR59 isoforms for binding to other binding partners.


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)

Association of the main human and murine PR72/B" isoforms with PP2AD. COS7 cells were transfected with GST, hPR130-GST, hPR72-GST, hPR70-GST, mG5PR/B"γ-GST, mPR59/B"δ1-GST, mPR59/B"δ2-GST and mPR59/B"δ3-GST. 48 h after transfection, a GST pull down assay was performed and binding of PR65/A and C subunits was evaluated via Western blotting using specific antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Association of the main human and murine PR72/B" isoforms with PP2AD. COS7 cells were transfected with GST, hPR130-GST, hPR72-GST, hPR70-GST, mG5PR/B"γ-GST, mPR59/B"δ1-GST, mPR59/B"δ2-GST and mPR59/B"δ3-GST. 48 h after transfection, a GST pull down assay was performed and binding of PR65/A and C subunits was evaluated via Western blotting using specific antibodies.
Mentions: With the exception of XN73 and mPR130/B"α3, all B" family members contain a conserved region and specific N- and C-termini. The conserved region, which contains two ASBDs and two EF-hand motifs, is necessary for PR65/A binding [3]. Since the first ASBD is not intact in mPR59/B"δ3 (Figure 2), we wondered whether this isoform is still able to bind PP2A. To this end, we made GST-fusion proteins of the main B" family members and overexpressed these in COS-7 cells. After a GST-pull down assay, we evaluated the binding of both the PR65/A and catalytic subunit of PP2A via Western blotting. As expected, all main B" isoforms (hPR130, hPR72, hPR70, mG5PR/B"γ, mPR59/B"δ1 and mPR59/B"δ2) bind PP2A (Figure 5), and are therefore genuine B" subunits. In contrast, mPR59/B"δ3 fails to bind PP2A, suggesting it is not a regulatory subunit of PP2A (Figure 5). Like XN73 and mPR130/B"α3, it might be involved in regulation of PP2A by competing with the other mPR59 isoforms for binding to other binding partners.

Bottom Line: The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.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.

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