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 α1, α2, β, and γ1, plus the Aα 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. 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 α1, α2, β, and γ1, plus the Aα 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. Protein phosphatase 1 (PP1) 1The abbreviations used are: PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; snRNP, small nuclear ribonucleoprotein particle; SAP145, spliceosome-associated protein 145; SAP155, spliceosome-associated protein 155; NIPP-1, nuclear inhibitor protein phosphatase 1; hnRNA, heterogeneous nuclear RNA; MALDI-TOF MS, matrix assisted laser desorption/ionization time-of-flight mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 2-ME, 2-mercaptoethanol; PMSF, phenylmethylsulfonyl fluoride; NE, nuclear extract; DIG-PP1, digoxygenin-3-O-methylcarbonyl-aminocaproic-acid-N-hydroxy-succinamide ester. 1The abbreviations used are: PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; snRNP, small nuclear ribonucleoprotein particle; SAP145, spliceosome-associated protein 145; SAP155, spliceosome-associated protein 155; NIPP-1, nuclear inhibitor protein phosphatase 1; hnRNA, heterogeneous nuclear RNA; MALDI-TOF MS, matrix assisted laser desorption/ionization time-of-flight mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 2-ME, 2-mercaptoethanol; PMSF, phenylmethylsulfonyl fluoride; NE, nuclear extract; DIG-PP1, digoxygenin-3-O-methylcarbonyl-aminocaproic-acid-N-hydroxy-succinamide ester. and 2A (PP2A) are highly conserved serine/threonine-specific protein phosphatases that have been identified in all eukaryotic species examined (1Cohen P. The structure and regulation of protein phosphatases..Annu. Rev. Biochem. 1989; 58: 453-508Google Scholar, 2Cohen P.T. Protein phosphatase 1—Targeted in many directions..J. Cell Sci. 2002; 115: 241-256Google Scholar). Dephosphorylation by PP1 is controlled by targeting or regulatory subunits that take PP1 to specific locations in the cell, potentially alter its phosphatase activity, and allow regulation by intra- or extracellular-derived signals (3Bollen M. Combinatorial control of protein phosphatase-1..Trends Biochem. Sci. 2001; 26: 426-431Google Scholar, 4Hubbard M.J. Cohen P. On target with a new mechanism for the regulation of protein phosphorylation..Trends Biochem. Sci. 1993; 18: 172-177Google Scholar, 5Ceulemans H. Stalmans W. Bollen M. Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution..Bioessays. 2002; 24: 371-381Google Scholar, 6Bollen M. Beullens M. Signaling by protein phosphatases in the nucleus..Trends Cell Biol. 2002; 12: 138-145Google Scholar). Biochemistry has shown that PP1 activity is highly enriched in the nucleus, and recent fluorescence microscopy studies with tagged versions of PP1 have dramatically illustrated this (7Trinkle-Mulcahy L. Sleeman J.E. Lamond A.I. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells..J. Cell Sci. 2001; 23: 4219-4228Google Scholar). PP1γ1 resides primarily in the nucleolar compartment, PP1α in the nucleoplasmic fraction, and PP1β in both nuclear compartments (7Trinkle-Mulcahy L. Sleeman J.E. Lamond A.I. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells..J. Cell Sci. 2001; 23: 4219-4228Google Scholar). Several nuclear PP1-targeting subunits have now been identified (6Bollen M. Beullens M. Signaling by protein phosphatases in the nucleus..Trends Cell Biol. 2002; 12: 138-145Google Scholar, 7Trinkle-Mulcahy L. Sleeman J.E. Lamond A.I. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells..J. Cell Sci. 2001; 23: 4219-4228Google Scholar, 8Sagara J. Higuchi T. Hattori Y. Moriya M. Sarvotham H. Shima H. Shirato H. Kikuchi K. Taniguchi S. ScapininA putative protein phosphatase-1 regulatory subunit associated with the nuclear nonchromatin structure..J. Biol. Chem. 2003; 278: 45611-45619Google Scholar, 9Kreivi J.P. Trinkle-Mulcahy L. Lyon C.E. Morrice N.A. Cohen P. Lamond A.I. Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity..FEBS Lett. 1997; 420: 57-62Google Scholar). The two most-abundant nuclear PP1 binding subunits, p99 or PNUTS and nuclear inhibitor PP1 (NIPP-1), are RNA-binding proteins that likely play a role in pre-mRNA splicing (9Kreivi J.P. Trinkle-Mulcahy L. Lyon C.E. Morrice N.A. Cohen P. Lamond A.I. Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity..FEBS Lett. 1997; 420: 57-62Google Scholar, 10Allen P.B. Kwon Y.G. Nairn A.C. Greengard P. Isolation and characterization of PNUTS, a putative protein phosphatase 1 nuclear targeting subunit..J. Biol. Chem. 1998; 273: 4089-4095Google Scholar, 11Boudrez A. Beullens M. Waelkens E. Stalmans W. Bollen M. Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1..J. Biol. Chem. 2002; 277: 31834-31841Google Scholar). Both proteins contain the PP1 binding motif R/K-V/I-X-F/W that was originally identified from studies on the glycogen and myosin PP1-targeting subunits (12Moorhead G.B.G. MacKintosh R.W. Morrice N. Gallagher T. Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin Sepharose affinity chromatography..FEBS Lett. 1994; 356: 46-50Google Scholar, 13Moorhead G.B.G. MacKintosh C. Morrice N. Cohen P. Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography..FEBS Lett. 1995; 362: 101-105Google Scholar, 14Johnson D.F. Moorhead G.B.G. Caudwell F.B. Cohen P. Chen Y.H. Chen M.X. Cohen P.T.W. Identification of protein phosphatase-1-binding domains on the glycogen and myofibrillar targeting subunits..Eur. J. Biochem. 1996; 239: 317-325Google Scholar, 15Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T.W. Cohen P. Barford D. Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1..EMBO J. 1997; 16: 1876-1887Google Scholar, 16Zhao S. Lee E.Y. A protein phosphatase-1-binding motif identified by the panning of a random peptide display library..J. Biol. Chem. 1997; 272: 28368-28372Google Scholar). This motif has been shown to be present in nearly all PP1-associating proteins. Due to the number of nuclear events controlled by phosphorylation/dephosphorylation, there undoubtedly exist many more, as yet unidentified proteins that target or localize nuclear protein phosphatases. Here, we have done an extensive examination of the mammalian nucleus for protein phosphatase-associated proteins by utilizing the protein phosphatase affinity matrix microcystin-Sepharose. This matrix has been used previously to successfully purify other protein phosphatase-targeting subunit complexes (12Moorhead G.B.G. MacKintosh R.W. Morrice N. Gallagher T. Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin Sepharose affinity chromatography..FEBS Lett. 1994; 356: 46-50Google Scholar, 13Moorhead G.B.G. MacKintosh C. Morrice N. Cohen P. Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography..FEBS Lett. 1995; 362: 101-105Google Scholar). 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 PP1 binding proteins exist in the nucleus than have been previously demonstrated. This work implicates protein phosphatases as regulators of many nuclear events. Unless stated otherwise, chemicals were from Other chemicals and were from the in PP1γ1 was and as previously P. Moorhead G.B.G. Protein phosphatases protein Biol. Chem. 2001; and for The was by S. and the protein A. an with activity serine/threonine protein phosphatases 1 (PP1) and 2A is highly for Lett. 1997; and on microcystin-Sepharose (12Moorhead G.B.G. MacKintosh R.W. Morrice N. Gallagher T. Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin Sepharose affinity chromatography..FEBS Lett. 1994; 356: 46-50Google Scholar). were and were and in of 2-ME, and two of were by for in a and in and with of were on of and in a for 1 The isolated nuclei were in plus and on for to nuclear The nuclei were by for in a and in A 2-ME, phenylmethylsulfonyl 1 and on the nuclei were and for in a the was and the nuclear in B 2-ME, PMSF, 1 and for in a this protein was with the and the with as and all the and the nuclear nuclear proteins protein liver were with 1 of microcystin-Sepharose matrix as (12Moorhead G.B.G. MacKintosh R.W. Morrice N. Gallagher T. Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin Sepharose affinity chromatography..FEBS Lett. 1994; 356: 46-50Google and previously in 1 the was with and protein was the were by for with and the was Protein was in plus and 1 in a and to in A control matrix was by with as by the the control matrix was the nuclear was in and microcystin and control Both were and and in to allow of proteins. A with of in the of of protein from the microcystin-Sepharose matrix and from the control with the affinity matrix was with and to with for and with further The was with a to and in to PP1 and catalytic subunits were from and were used The was from and used a to ZAP was by the ZAP peptide to and as in D. A. Moorhead G.B.G. of of the nuclear protein phosphatase in rat liver Cell Biol. 2002; and were affinity on a peptide affinity D. A. Moorhead G.B.G. of of the nuclear protein phosphatase in rat liver Cell Biol. 2002; Scholar). to the myosin-targeting subunits and were by P. Cohen of Other were by the and used as in the and p99 Trinkle-Mulcahy and A. Lamond (7Trinkle-Mulcahy L. Sleeman J.E. Lamond A.I. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells..J. Cell Sci. 2001; 23: 4219-4228Google Scholar, 9Kreivi J.P. Trinkle-Mulcahy L. Lyon C.E. Morrice N.A. Cohen P. Lamond A.I. Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity..FEBS Lett. 1997; 420: 57-62Google U5 small nuclear ribonucleoprotein p116 and and P. J. conserved U5 protein is a related to the J. 1997; 16: Scholar, J. P. S. E. The U5 protein and its in are of the protein of putative RNA J. 1996; U2 snRNPs spliceosome-associated protein and SAP155 L. L. Y. of a protein spliceosomal proteins and Biol. Scholar, C. K. W. E. of spliceosomal protein with splicing 1998; 12: and myosin heavy C. A. A. studies with to from the and of were by for to in in and were for 1 and for with and with the were using the ZAP of ZAP or was with of and as Moorhead G.B.G. and of the putative of 2002; Scholar). as plus was with of Sepharose for with 1 of plus and proteins were with of SAP155, of was with of Sepharose and was with of Sepharose for with 1 of plus and proteins were with of PP1γ1 was labeled with digoxygenin-3-O-methylcarbonyl-aminocaproic-acid-N-hydroxy-succinamide as G.B.G. MacKintosh C. Morrice N. Cohen P. Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography..FEBS Lett. 1995; 362: 101-105Google and used the were on to and were for and for with and with the with on the PP1 binding a peptide was the PP1 binding of M. A. J. S. Stalmans W. Bollen M. of nuclear protein phosphatase-1 regulation by Biol. Chem. and The control peptide was was with an PP1 binding to its targeting subunits M.P. Johnson D.F. Moorhead G. Cohen P.T.W. Cohen P. Barford D. Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1..EMBO J. 1997; 16: 1876-1887Google Scholar, M. A. J. S. Stalmans W. Bollen M. of nuclear protein phosphatase-1 regulation by Biol. Chem. Scholar). The peptide on the putative PP1 binding site of ZAP was used in PP1 as were by of using on an peptide The were by and the peptide and was by liquid chromatography and were on with and the was The protein was with of with the in D. Moorhead G.B.G. Purification and of type 1 protein phosphatase 2001; Scholar). were on a laser desorption/ionization time-of-flight mass with as the The mass was in the and was mass The peptide were the and using the of on a all the mass was for of or there was an in the of a protein liquid chromatography mass was The was on to a with in and to an The was with a and the was to a mass The peptide by the were using The were using the the have previously shown that the nuclear used here results in highly nuclei that are with cytosolic proteins D. A. Moorhead G.B.G. of of the nuclear protein phosphatase in rat liver Cell Biol. 2002; Scholar). we have the isolated nuclear and cytosolic with the cytosolic protein and confirmed that the nuclear is of cytosolic the that nuclear PP1 regulatory subunits proteins from isolated nuclei by and were on to a and with labeled This has previously been established as a to identify putative PP1 binding proteins G.B.G. MacKintosh C. Morrice N. Cohen P. Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography..FEBS Lett. 1995; 362: 101-105Google Scholar, 14Johnson D.F. Moorhead G.B.G. Caudwell F.B. Cohen P. Chen Y.H. Chen M.X. Cohen P.T.W. Identification of protein phosphatase-1-binding domains on the glycogen and myofibrillar targeting subunits..Eur. J. Biochem. 1996; 239: 317-325Google Scholar). The that many more nuclear PP1 binding proteins exist than have been previously (9Kreivi J.P. Trinkle-Mulcahy L. Lyon C.E. Morrice N.A. Cohen P. Lamond A.I. Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity..FEBS Lett. 1997; 420: 57-62Google Scholar). this protein phosphatases to microcystin were on a microcystin affinity and work (9Kreivi J.P. Trinkle-Mulcahy L. Lyon C.E. Morrice N.A. Cohen P. Lamond A.I. Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity..FEBS Lett. 1997; 420: 57-62Google the PP1 binding proteins of and and were to be and A of the other putative PP1 binding proteins The binding is to the nuclear that a of the nuclear PP1 binding proteins on the microcystin The binding of PP1 to proteins be by of a peptide the PP1 binding site of the known PP1 regulatory subunit and the key binding of the M. A. J. S. Stalmans W. Bollen M. of nuclear protein phosphatase-1 regulation by Biol. Chem. to an this interaction and that proteins are nuclear PP1 binding proteins and to the of proteins. with results affinity chromatography results in a of binding to the support a control for we the chromatography by the nuclear proteins two and the microcystin and matrix in proteins were to the and proteins were by A in to more protein binding and from the microcystin matrix compared with the control shown in several proteins are in on both and be from further the of proteins between the and and the These proteins were identified as and and the putative The proteins on microcystin-Sepharose were on a and the of proteins PP1 binding proteins is compared in A and for of the are shown to of binding proteins for PP1 and and the with the protein that with PP1 to be identified This that several proteins on the were PP1 binding proteins. that to PP1 also be PP1 binding proteins to in an overlay also be binding proteins or protein of protein phosphatase is that are subunits of protein phosphatases we catalytic subunits of this of phosphatases in the nuclear microcystin-Sepharose an to further proteins were to the matrix we a was that this the catalytic subunit that is with affinity to this we have been in the to from the (12Moorhead G.B.G. MacKintosh R.W. Morrice N. Gallagher T. Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin Sepharose affinity chromatography..FEBS Lett. 1994; 356: 46-50Google Scholar). The proteins were with and are shown in The is a protein of all the regulatory subunits from the as the is PP1 by MALDI-TOF be by in MALDI-TOF and as the to identify the proteins from the microcystin and the results are in and in MALDI-TOF confirmed that of the mammalian PP1 are present in the to α2, β, and were in the mass is that is present in the nucleus, is PP1γ1 and in a in their The peptide to was present in the mass to the Aα subunit were identified with several proteins to the B and of the number and of the B and B′ subunits, we and identify to were Other protein identified include the myosin heavy chain, the U2 snRNPs SAP145, SAP155, the U5 snRNPs p116 and p200, a putative RNA binding protein binding protein and p99, NIPP-1, and a putative related to have used to the specific of of proteins to the microcystin proteins enriched on microcystin-Sepharose chromatography and identified by mass mass protein heavy binding binding protein protein Aα ribonucleoprotein in a new of the proteins identified by mass spectrometry were of in to other as p99 or further a specific with the microcystin Western were with from control and affinity matrix were for PP1, p99, and to the of the microcystin that the splicing factors U5 p116 and and U2 snRNPs SAP145 and SAP155 with a protein phosphatase or that a protein phosphatase with the splicing with affinity to allow of the that splicing factors with the control matrix are enriched on microcystin-Sepharose. The of of splicing factors that with the affinity matrix is of the of splicing present in a nuclear the interaction of the U2 snRNPs with a protein we a using the SAP155 This that PP1, was in a with the U2 SAP155 The targeting of a protein phosphatase to the splicing is with results that of the splicing are by The was identified by as myosin to the control this was enriched to on the microcystin matrix for several of this we the nuclear and microcystin and with the myosin phosphatase regulatory proteins and for the subunit were in the nuclear or A of and a of were in nuclear and the microcystin-Sepharose for the the The is shown is likely the the to PP1 (12Moorhead G.B.G. MacKintosh R.W. Morrice N. Gallagher T. Mackintosh C. Purification of type 1 protein (serine/threonine) phosphatases by microcystin Sepharose affinity chromatography..FEBS Lett. 1994; 356: 46-50Google Scholar). The putative RNA binding protein ZAP was also shown to with the affinity matrix further an interaction between PP1 and we ZAP Western analysis of nuclear and cytosolic with this an the as the identified as ZAP in and an protein of Both were localized to the nucleus with the both the and were by Western with the the also confirmed that PP1, a with ZAP Far Western show that the and ZAP proteins also have the to PP1 in an form a interaction with PP1, and this be with the ZAP peptide the putative PP1 binding site with the U2 A nuclear was with or and of proteins by to and with SAP155, PP1, and The labeled of nuclear of the ZAP were affinity and used 1 to cytosolic and nuclear by and to A nuclear was with and proteins by to and with PP1 and The was also with labeled PP1 to that the ZAP the to PP1 and this be by of the peptide from The nucleus is a and highly protein a key role in a of in the mammalian nucleus, yet is known the nuclear protein phosphatases. have the of the protein phosphatase affinity matrix microcystin-Sepharose, the PP1 overlay and mass spectrometry to identify proteins and phosphatase complexes of the mammalian this we have isolated rat liver and that many more PP1 binding proteins are present in the nucleus previously These proteins on microcystin-Sepharose and several were utilizing microscopy and other studies have shown that the PP1 isoforms β, and are present in the nucleus F.B. E. of protein phosphatase-1 and isoforms both and in mammalian cells..J. Cell Biol. 1998; their between and or Trinkle-Mulcahy (7Trinkle-Mulcahy L. Sleeman J.E. Lamond A.I. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells..J. Cell Sci. 2001; 23: 4219-4228Google have shown PP1γ1 and to be highly enriched in the and using PP1 proteins. Our mass spectrometry that of the mammalian PP1 are present in the The between PP1γ1 and is a on the that is by splicing. The peptide to was identified in mass the peptide to The Aα and is likely a of B and B′ subunits of were also present in the have identified a previously as as a new PP1-targeting This protein on microcystin-Sepharose, to PP1 in an overlay be by a peptide the PP1 binding 1 and and has a PP1 binding motif its This interaction was by ZAP from a nuclear and that PP1, and the ZAP PP1 in the overlay A of established PP1 binding proteins and their show that a of binding to the This in with from panning a random peptide with PP1 S. Lee E.Y. A protein phosphatase-1-binding motif identified by the panning of a random peptide display library..J. Biol. Chem. 1997; 272: 28368-28372Google also a for other is an two to the and a or between the and A of the The putative PP1 binding site in ZAP all this criteria and we that PP1 with ZAP the site the peptide on this site the binding of PP1 to ZAP with the that this is the PP1 binding this motif resides the of the protein and likely is on the protein and The of ZAP in the nucleus is the and motif with the protein that ZAP has that display to RNA the protein is an binding and and several pre-mRNA splicing The ZAP protein an of and domains by an and have been to be present in RNA binding proteins associated with pre-mRNA splicing analysis of the 2002; Scholar, J. Lamond A.I. M. analysis of the 2002; 12: including and U2 all of were identified as in this a of ZAP to RNA ZAP has in many splicing a recent of the spliceosomal proteins identify ZAP analysis of the 2002; Scholar, J. Lamond A.I. M. analysis of the 2002; 12: Scholar). work the role of targeting PP1 to this nuclear The splicing of is by a nuclear to as the is of proteins and small nuclear and analysis of the 2002; Scholar, J. Lamond A.I. M. analysis of the 2002; 12: Scholar). is a of spliceosomal a of factors from a nuclear known as The of has that the is to and many protein in K. J. nuclear ribonucleoprotein U2 proteins and has a structure splicing Biol. 1993; Scholar). has protein with and of the J.E. Cohen P. Lamond A.I. protein phosphatases are for both catalytic of pre-mRNA Scholar, J.E. Cohen P.T. Lamond A.I. of mammalian by a protein J. 1994; Scholar, T. Protein and the nuclear of pre-mRNA Cell Biol. 1997; Scholar, M. Bollen M. The protein phosphatase-1 is also a splicing in a of Biol. Chem. 2002; 277: with and the protein phosphatases and Western analysis confirmed the results that U5 snRNPs p116 and and U2 snRNPs SAP145 and SAP155 are on the microcystin affinity The U2 also to be enriched compared with the control of the nuclear of factors was to the with the that a of factors are in splicing and that protein phosphatases with the a with SAP155 and that PP1 is in this This is with a recent was that the of the nuclear PP1-targeting subunit with SAP155 in a M. Bollen M. The protein phosphatase-1 is also a splicing in a of Biol. Chem. 2002; 277: Scholar). SAP155 is in functional and this with or the splicing of the U2 C. K. W. E. of spliceosomal protein with splicing 1998; 12: Scholar). This that a take is likely that the U2 snRNPs and a of the splicing were on affinity matrix to the of with The that a of splicing factors to be enriched on the phosphatase affinity matrix the of protein phosphatases with the specific of and is also likely that this is to the of the splicing K. J. nuclear ribonucleoprotein U2 proteins and has a structure splicing Biol. 1993; Scholar). Our work the that protein phosphatases are of the splicing mass analysis of splicing and spliceosomal proteins from the to the of protein phosphatase subunits, with the of analysis of the 2002; Scholar). This is likely to the that protein phosphatase have been used of the Here, we have the nuclear with a phosphatase inhibitor to a This all protein phosphatase activity and the of the PP1 or has been isolated with spliceosomal yet we have several splicing with protein that phosphatases with the splicing in a work is to specific for protein phosphatases in splicing events and to protein phosphatase regulatory subunits other than target to the splicing The or are a of to play a role in the of W. P. J. M. The of a new of the on and Biol. Chem. 2002; 277: Scholar). there is the interaction of of with a protein phosphatase phosphatase or the specific to the is that this to on matrix and and is associated with a protein phosphatase or in a phosphatase be an in with as a in has been localized to the nucleus G. L. P. A. C. G. P. for the presence of myosin in the Biol. Chem. 1997; 272: to this is the of a myosin PP1-targeting subunit in the nucleus of the and likely the of myosin on work is to the role of PP1 to myosin in the were to all proteins that to be enriched on the microcystin matrix are associated or These proteins further Our overlay several more PP1 binding proteins with between and that are present in the mass spectrometry the protein to be Western analysis that this PP1 binding protein is we have been to identify other proteins and further work is to their that and likely more, PP1 binding proteins are present in the Due to the binding of protein and likely complexes to Sepharose we have the of the of microcystin-Sepharose to purify PP1 complexes from nuclear of the be to and further complexes to identify PP1-targeting Cohen for on the The catalytic subunit of protein was the of and S. P.

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