Scapinin, a Putative Protein Phosphatase-1 Regulatory Subunit Associated with the Nuclear Nonchromatin Structure

Abstract

It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654–1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human. It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654–1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human. Protein phosphatase-1 (PP1) 1The abbreviations used are: PP1, protein phosphatase-1; DAPI, 4′,6-diamidino-2-phenylindole; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; GST, glutathione S-transferase; HA, hemagglutinin; hnRNP, heterogeneous nuclear ribonucleoprotein; IF, intermediate filament; MES, 4-morpholineethanesulfonic acid; NM, nuclear matrix; P1, particulate; P2, chromatin-depleted fraction; PIPES, 1,4-piperazinediethanesulfonic acid; PP1c, α-catalytic subunit of PP1; RA, all-trans-retinoic acid; RACE, rapid amplification of cDNA ends; RT, reverse transcription; Sol, soluble; TBS, Tris-buffered saline. is a major eukaryotic serine/threonine protein phosphatase regulating diverse cellular processes such as muscle contraction, glycogen metabolism, RNA processing, and neuronal signaling (1Cohen P. Trends Biochem. Sci. 1992; 17: 408-413Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 2Bollen M. Stalmans W. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar, 3Boudrez A. Beullens M. Groenen P. Van Eynde A. Vulsteke V. Jagiello I. Murray M. Krainer A.R. Stalmans W. Bollen M. J. Biol. Chem. 2000; 275: 25411-25417Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The catalytic subunit of PP1 binds to regulatory subunits that are critical for substrate specificity and spatial control of PP1 within the cell (4Cohen T.P. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). It is thought that the nuclear nonchromatin structures, such as the nuclear matrix (NM) and nuclear lamina, are implicated not only in the spatial segregation of chromatins but also in the regulation of various nuclear processes associated with them (5Berezney R. Coffey D.S. Science. 1975; 189: 291-293Crossref PubMed Scopus (350) Google Scholar, 6Mirkovitch J. Mirault E. Laemmli U.K. Cell. 1984; 39: 223-232Abstract Full Text PDF PubMed Scopus (858) Google Scholar, 7Ma H. Siegel A.J. Berezney R. J. Cell Biol. 1999; 146: 531-541Crossref PubMed Scopus (146) Google Scholar). However, the regulatory mechanisms of the nuclear nonchromatin structure are not fully understood. The α-catalytic subunit of PP1 is associated with NM during interphase, and PP1 is a mitotic lamin phosphatase (8Andreassen P.R. Lacroix F.B. Villa-Moruzzi E. Margolis R.L. J. Cell Biol. 1998; 141: 1207-1215Crossref PubMed Scopus (173) Google Scholar, 9Thompson L.J. Bollen M. Fields A.P. J. Biol. Chem. 1997; 272: 29693-29697Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Recruitment of PP1 to the nuclear envelope by a PP1-binding protein, protein kinase A-anchoring protein AKAP-149, is a prerequisite for nuclear lamina assembly at the end of mitosis (10Steen R.L. Martins S.B. Tasken K. Collas P. J. Cell Biol. 2000; 150: 1251-1261Crossref PubMed Scopus (136) Google Scholar). Identification of PP1-binding proteins associated with the nuclear nonchromatin structure will provide insights into the regulatory roles of PP1 in the nuclear structure and gene expression. Aberrant nuclear structures are hallmarks of neoplastic transformation (11Stein G.S. Montecino M. van Wijnen A.J. Stein J.L. Lian J.B. Cancer Res. 2000; 60: 2067-2076PubMed Google Scholar). Nuclear lamins alter during differentiation and transformation (12Kaufmann S.H. Cancer Res. 1992; 52: 847-853Google Scholar, 13Oguchi M. Sagara J. Matsumoto K. Saida T. Taniguchi S. Br. J. Dermatol. 2002; 147: 853-858Crossref PubMed Scopus (23) Google Scholar). Many reports have described striking alterations in the NM proteins induced by transformation (14Partin A.W. Getzenberg R.H. CarMichael M.J. Vindivich D. Yoo J. Epstein J.I. Coffey D.S. Cancer Res. 1993; 53: 744-746PubMed Google Scholar, 15Khanuja P.S. Lehr J.E. Soule H.D. Gehani S.K. Noto A.C. Choudhury S. Chen R. Pienta K.J. Cancer Res. 1993; 53: 3394-3398PubMed Google Scholar, 16Keesee S.K. Meneghini M.D. Szaro R.P. Wu Y.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1913-1916Crossref PubMed Scopus (86) Google Scholar, 17Bidwell J.P. Fey E.G. van Wijnen A.J. Penman S. Stein J.L. Lian J.B. Stein G.S. Cancer Res. 1994; 54: 28-32PubMed Google Scholar, 18Getzenberg R.H. Konety B.R. Oeler T.A. Quigley M.M. Hakam A. Becich M.J. Bahnson R.R. Cancer Res. 1996; 56: 1690-1694PubMed Google Scholar). The mechanisms and physiological importance of this phenomenon are not fully understood, and identification of the alterations may provide more insight into differentiation and transformation. HL-60 cells have been used as an in vitro model of differentiation because various compounds can cause differentiation of these cells into granulocytes or monocytes (19Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2458-2462Crossref PubMed Scopus (1428) Google Scholar, 20Breitman T.R. Selonick S.E. Collins S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2936-2940Crossref PubMed Scopus (1864) Google Scholar). In this study, we analyzed differentiation-associated alterations of the chromatin-depleted nuclear structure, referred to as the NM-intermediate filament (IF) scaffold (21Fey E. Krochmalnic G. Penman S. J. Cell Biol. 1986; 102: 1654-1665Crossref PubMed Scopus (331) Google Scholar), of HL-60 cells. We then identified a novel protein, scapinin, which was tightly attached to the NM-IF scaffold of the nucleus and down-regulated by differentiation in HL-60 cells. Furthermore, we identified PP1 as a scapinin-binding protein by the yeast two-hybrid assay. Cell Culture—HL-60 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. To induce differentiation, HL-60 cells (2 × 105/ml) were treated with 100 nm all-trans-retinoic acid (RA). Maximum reduction of nitro blue tetrazolium (a differentiation marker) occurred after treating HL-60 cells with RA for 5 days. HL-60 cells (5 × 105/ml) were also treated with 10 nm phorbol 12-myristate-13-acetate or 1.2% dimethyl sulfoxide. U937, K562, and Jurkat cells were cultured in RPMI 1640, and HeLa, WM35, SKNSH, and GOTO cells were cultured in Dulbecco's modified minimal essential medium. All media were supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin. Preparation of the NM-IF Scaffold and IF Fraction of HL-60 Cells—The NM-IF scaffold was prepared as described previously (21Fey E. Krochmalnic G. Penman S. J. Cell Biol. 1986; 102: 1654-1665Crossref PubMed Scopus (331) Google Scholar). Briefly, the cells were suspended in 0.5% Triton X-100 and cytoskeleton buffer (10 mm PIPES, pH 6.8, 100 mm KCl, 300 mm sucrose, 3 mm MgCl2,1mm EGTA, 4 mm vanadyl riboside complex) on ice for 5 min before separating the particulate (P1) and soluble (Sol) fractions by centrifugation at 600 × g for 5 min. The P1 fraction was incubated with 0.15 mg/ml DNase I at 23 °C for 30 min. Ammonium sulfate (1 m) was added to obtain a final concentration of 0.25 m, and the chromatin-depleted fraction (P2) was then collected by centrifugation. The P2 fraction was further extracted with a 2 m NaCl solution. Fey et al. (21Fey E. Krochmalnic G. Penman S. J. Cell Biol. 1986; 102: 1654-1665Crossref PubMed Scopus (331) Google Scholar) referred to this final particulate fraction as the NM-IF scaffold. The NM-IF scaffold was separated into the NM and IF fractions as described previously (23Fey E.G. Penman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 85: 121-125Crossref Scopus (278) Google Scholar). The NM-IF scaffold was dissolved in an 8 m urea solution containing 20 mm MES, pH 6.6, 0.1 mm MgCl2, 1 mm EGTA, and 1% 2-mercaptoethanol. After ultracentrifugation at 100,000 × g for 1 h, the supernatant was dialyzed against a buffer containing 25 mm imidazole, pH 7.1, 150 mm KCl, 5 mm MgCl2 and 0.125 mm EGTA. The reassembled (IF fraction) and soluble (NM) proteins were separated by centrifugation at 10,000 × g for 15 min. To release RNA-containing matrix, the NM-IF scaffold was digested with RNase A in cytoskeleton buffer containing protease inhibitors; NaCl was then added to a final concentration of 2 m from a stock solution of 4 m NaCl in cytoskeleton buffer. 1 mm dithiothreitol was included in all solutions used in preparation of the NM-IF scaffold and RNase A digestion. RNase A (Sigma) was for 20 min to DNase and protease The of RNase A was by the of heterogeneous nuclear was from of a NM-IF scaffold proteins from proliferating or HL-60 cells were with and to of After 3 were with of the NM-IF scaffold proteins with 3 after the the cells were with and in K. Taniguchi S. J. S. H. A. T. Sagara J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The NM-IF scaffold proteins from proliferating or HL-60 cells were suspended in by at a final concentration of 10 and was added to assay at 0.1 The a in an assay with the NM-IF scaffold proteins of proliferating cells but was in that of cells. of Scapinin cDNA was from RNA of HL-60 cells and as described K. Taniguchi S. J. S. H. A. T. Sagara J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). were by with and reverse The was into the and to The of the was using a to the The of the was by the The was into the and The of and were by using a a reverse a a RNA of HL-60 and a RNA The of and were separated by 1% into and and was as described previously K. Taniguchi S. J. S. H. A. T. Sagara J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). was as HL-60 cells and the NM-IF scaffold of HL-60 cells were on using with for 15 and with containing 1% bovine and fetal bovine at for 15 min. The cells were incubated for 1 at with from an After with TBS, the cells were incubated with fluorescein or for 30 min. The were with at 1 μg/ml for 5 min. To the of the NM-IF were incubated for 10 min with a at 1 The was using a After with TBS, the were using a HL-60 cells were also with against the splicing leukemia protein, or proliferating cell nuclear of cells were separated by in 1% containing and then with A of scapinin cDNA by using the and reverse was used as the The was using the and and with the in 5 × at RNA were also of for and were into and to cells using the The of and were by using the to and The were then into The were into cells on were analyzed with the two-hybrid to the The and of were into the of with the The yeast was transformed by the and by containing the human cDNA in and media were analyzed for were to the yeast were incubated at 30 °C in 5 of the medium. 2 of the was added to 8 of and incubated at 30 °C the A 600 After with buffer mm mm 10 mm KCl, 1 mm of the was in 0.1 of buffer. were by and with of buffer containing The was then by the of of 4 mg/ml (Sigma) and incubated at 30 The A was after the with of 1 m activity was from the of A and A 600 using 1 is the of the was as as and as α-catalytic subunit of PP1 was expressed as a protein using the as described H. K. A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). 5 × cells were with or and of of was used for the the control was added to the to cells were in buffer (10 mm pH 100 mm KCl, 10% 1% Triton X-100, and protease with the of and cell was by centrifugation at × g for 20 min. The cell was into two and incubated with or for 3 were collected by centrifugation × 1 and in buffer. were analyzed by using or PP1 by was from HL-60 cells. The NM-IF scaffold was dissolved in buffer 0.5% 1% 150 mm and mm pH and the was incubated with for 3 at 4 After with buffer concentration was to scapinin was in buffer. scapinin was separated by and then on was as described H. H. G. T. M. K. 2000; 39: PubMed Scopus Google Scholar). Briefly, were incubated with or μg/ml (Sigma) in (10 mm pH 150 mm and containing mm for 4 at 4 was by with for 1 by with for 30 min. PP1 were expressed as proteins with The cDNA of scapinin was by using a to which or were After with and the was into and with and proteins were in and with The catalytic subunit of PP1 was from and PP1 activity was as described H. H. G. T. M. K. 2000; 39: PubMed Scopus Google Scholar). mm 0.15 mm 15 mm mg/ml bovine 5 mm 10 and 100 PP1 with various of or of Scapinin with the Nuclear Scaffold in HL-60 against NM-IF scaffold proteins of We a an which two with of and were in the NM-IF scaffold The NM-IF scaffold was separated further into the NM and IF fractions by in an 8 M urea solution before in a physiological solution as described previously (23Fey E.G. Penman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 85: 121-125Crossref Scopus (278) Google Scholar). proteins (IF fraction) were separated from soluble proteins fraction) by centrifugation The two were in the IF The NM-IF scaffold a filament of nuclear RNA and (21Fey E. Krochmalnic G. Penman S. J. Cell Biol. 1986; 102: 1654-1665Crossref PubMed Scopus (331) Google Scholar). To the of scapinin to the filament we incubated the NM-IF scaffold of HL-60 cells with RNase RNase NM-IF scaffold was then and analyzed We to the of the RNase A digestion. of the scapinin was from the NM-IF scaffold by RNase A digestion. However, of the of scapinin was to RNase A digestion. was by such The that scapinin is or associated with the filament The the of HL-60 cells also of the scapinin with the NM-IF scaffold of the nucleus These results that the scapinin is a of the nuclear scaffold of HL-60 cells. a of scapinin in the nucleus of HL-60 cells We the of scapinin with that of nuclear proteins and that scapinin was not with the splicing leukemia protein or proliferating cell nuclear and not of of a cDNA by of a cDNA and then a cDNA by and The cDNA a novel of 518 amino acids of the protein and of scapinin nuclear at To the we using that were the and a we two that two splicing of 448 (scapinin-S) and 518 amino A of the human that the scapinin gene was in and of 5 amino acids in revealed that and and which with the of HL-60 cells of that scapinin was to proteins that have been only from of cDNA that scapinin is a of a novel protein family of protein but to such as protein and subunit All of these proteins are cellular proteins and have to scapinin, but the of were of the proteins Scapinin in Cell and that scapinin was expressed in HL-60 and GOTO but not in Jurkat or cells In human scapinin were in the the was for scapinin were in the and not The of scapinin in the was not We then analyzed human in I, of a of scapinin were from cDNA The scapinin were from of or or of scapinin Scapinin were by of the human using cDNA as a of cDNA of of which are of also are into and of which are of also are into and in a was the major and the in and GOTO as in HL-60 cells. with the that scapinin was in the of and GOTO cells not of Scapinin in HL-60 by of and that scapinin was by RA of HL-60 cells Furthermore, of scapinin was induced by with phorbol 12-myristate-13-acetate and dimethyl that the for scapinin in the nucleus were in HL-60 cells treated with compounds not of the of two splicing variants of scapinin were expressed in cells using the and the cells or were extracted with buffers containing and as in The expressed and were in the NM-IF scaffold The NM-IF from or cells were digested further with RNase RNase insoluble NM-IF were then analyzed as in of and was from the NM-IF scaffold by RNase A was by RNase A digestion. To the of the two variants of scapinin, we expressed them as proteins in cells was in the is with the of HL-60 cells However, the expressed in cells was in in to the To the of GFP, we expressed using the and the cells with the as in Furthermore, we the to various cell WM35, SKNSH, and GOTO and the In all cell and the nucleus were with the not We that because is the in HL-60 we the of in HL-60 cells. are to this Identification of PP1 as a in We then for scapinin-binding proteins by the yeast two-hybrid assay to the the α-catalytic subunit of PP1 were as for scapinin-binding To scapinin with in and scapinin or were in cells and to a assay with and of to scapinin in the cells. We of scapinin with by using as a in scapinin from HL-60 cells bound with We was in the NM-IF scaffold of HL-60 in with with that was not in the NM-IF scaffold of HL-60 cells and that the concentration of in the NM-IF scaffold fraction was the with that of the cell not We that the extraction used in this from It is that in of regulatory subunits with are and are by is that only of the of scapinin in the NM-IF scaffold with in HL-60 cells. will to this To the of scapinin with PP1c, of scapinin were to the yeast two-hybrid assay. revealed that the amino of scapinin was the PP1 In this a sequence which is in PP1-binding is It is that PP1-binding proteins PP1 of scapinin were expressed as proteins and to PP1 inhibited PP1 and a 10 amino acids from the inhibited PP1 These results that scapinin binds to and PP1 and that is for PP1 and PP1 inhibiting Scapinin the presence of scapinin in and were In the and were them of the the presence of an in the The is named after the amino acids and that it The of the is Furthermore, all of these proteins have a for which the after RNA splicing These results suggest that scapinin and are of a novel family that are from to We therefore this family the scapinin Scapinin was associated with the NM-IF scaffold and and in the IF fraction A that scapinin but to various of cellular protein and subunit is a protein with an essential in mitosis Chen J. Cell Biol. 1993; PubMed Scopus Google Scholar). is to and a in the of the structure of the and in the Chen J. Cell Sci. Google Scholar). is a IF protein P.S. M.J. J. PubMed Scopus Google Scholar). and also to and The results of the and the and suggest that scapinin may as a of the nucleus in HL-60 cells and of cells. The NM-IF scaffold a nuclear filament of RNA and which is from the nuclear insoluble scaffold by RNase (21Fey E. Krochmalnic G. Penman S. J. Cell Biol. 1986; 102: 1654-1665Crossref PubMed Scopus (331) Google Scholar). of the scapinin was from the NM-IF scaffold by RNase but the was to RNase A and was by such These results suggest that a structure is by with the nuclear and is and by the of nuclear of the scapinin is associated with the nuclear Scapinin RNA in and are to the scapinin and the nuclear of the regulatory subunits to PP1 a the However, putative regulatory subunits not the but tightly to PP1 by of at (4Cohen T.P. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar, M. Trends Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). Scapinin not an PP1 and that the of amino acids is for the PP1 and The that a critical for PP1 amino acids In of 10 amino acids from the the PP1 inhibiting activity These results suggest that in the of scapinin are for the PP1 PP1 roles in the regulation of various cellular processes in the nucleus and the It is thought that the nuclear nonchromatin structures, such as the NM and nuclear lamina, are implicated not only in the spatial segregation of chromatins but also in the regulation of various nuclear processes associated with However, the regulatory mechanisms of the nuclear scaffold are not fully understood. Scapinin is a putative PP1 regulatory subunit associated with a nuclear nonchromatin structure, a regulatory of scapinin in the further on scapinin may provide an insight into the regulatory roles of the nuclear scaffold leukemia HL-60 which in can induced to into and granulocytes or monocytes by with a of compounds (19Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2458-2462Crossref PubMed Scopus (1428) Google Scholar, 20Breitman T.R. Selonick S.E. Collins S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2936-2940Crossref PubMed Scopus (1864) Google Scholar). Scapinin was down-regulated in HL-60 cells during differentiation induced by RA, dimethyl and phorbol of was not in HL-60 cells during The of scapinin occurred at the and suggest that scapinin is to of and that it is not a gene 4 and scapinin may to transformed or immature phenotypes of of cells. In scapinin is in the a in the that was in the but was also in in to the nucleus The of the for to with the on HL-60 cells that was the with in HL-60 cells a for this is that the only the of on GOTO and in which was the as in HL-60 also nuclear of scapinin not are to the for in the and the of this amino acids containing a sequence It is that as which are implicated in various cellular processes such as cell and gene M. T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Trends Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Scapinin family proteins also that or involved in identification of proteins to the sequence may and Scapinin is a of a novel protein family from to These scapinin family proteins to as a with the of a sequence of amino acids The is a in and and an of D. P.S. E. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, V. M. D. D. J. PubMed Scopus Google Scholar, T. R. M. T. R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). These proteins have a that is a putative to involved in The of amino to which the putative PP1 of scapinin was is scapinin family proteins and the that the scapinin family a novel PP1-binding protein are to this We are to K. of for in the of this and to T. and K. for on the yeast two-hybrid assay.