Proteomic characterization of protein phosphatase complexes of the mammalian nucleus.
Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts. Microcystin-Sepharose chromatography and mass spectrometry were utilized to affinity purify and identify protein phosphatase-associated proteins from isolated rat liver nuclei. Far Western analysis with labeled protein phosphatase 1 (PP1) showed that many more PP1 binding proteins exist in the nucleus than were previously demonstrated. Mass spectrometry confirmed the presence in the nucleus of the mammalian PP1 isoforms alpha1, alpha2, beta, and gamma1, plus the Aalpha and several of the B and B' subunits that are complexed to PP2A. Other proteins enriched on the microcystin matrix include the spliceosomal proteins known as the U2 snRNPs SAP145 and SAP155 and the U5 snRNPs p116 and p200, myosin heavy chain, and a nuclear PP1 myosin-targeting subunit related to M110. The putative RNA binding protein ZAP was also established as a nuclear PP1 binding protein using the criteria of co-purification with PP1 on microcystin-Sepharose, co-immunoprecipation, binding PP1 in an overlay assay, and presence of a putative PP1 binding site (KKRVRWAD). These results further support a key role for protein phosphatases in several nuclear functions, including the regulation of pre-mRNA splicing.
Tran
Hue T
HT
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Ulke
Annegret
A
Morrice
Nick
N
Johannes
Christine J
CJ
Moorhead
Greg B G
GB
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002847:Chromatography, Agarose; D008297:Male; D013058:Mass Spectrometry; D052998:Microcystins; D009687:Nuclear Proteins / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D040901:Proteomics; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012685:Sepharose / Q000378:metabolism
D052998:Microcystins; D009687:Nuclear Proteins; D010456:Peptides, Cyclic; D012322:RNA Precursors; C078588:microcystin; D012685:Sepharose; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1