Abstract
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.
Highlights
Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts
Using a combination of affinity chromatography and mass spectrometry, we have identified many new proteins localized to protein phosphatase complexes, and here we show many more phosphatase 1 (PP1) binding proteins exist in the nucleus than have been previously demonstrated
Further Nuclear PP1 Binding Proteins Are Revealed—We have previously shown that the nuclear isolation technique used here results in highly pure nuclei that are not contaminated with cytosolic proteins [19]
Summary
Materials—Unless stated otherwise, chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Other chemicals and reagents were obtained from the companies indicated in parentheses. Human PP1␥1 was expressed and purified as described previously [17] and dialyzed into 25 mM NaHCO3, pH 7.5, 200 mM KCl, 50% (v/v) glycerol, 0.1% (v/v) 2-mercaptoethanol (2-ME) for storage. The human PP1␣1 clone was kindly provided by S. Shenolikar (Duke University), transfected into Escherichia coli BL21 (DE3), and the protein expressed [18] and purified on microcystin-Sepharose [12]
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