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• W2087422952 abstract "Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr320, which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTFmotif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes. Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr320, which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTFmotif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes. Reversible phosphorylation of proteins is the major mechanism for control of myriad functions in eukaryotic cells. Kinases and phosphatases catalyze the opposing reactions, and we have been interested in how these activities are reciprocally regulated to produce abrupt and transient changes in the phosphorylation state of target proteins. Protein phosphatase-1 (PP1) 1The abbreviations used are: PP1, protein phosphatase 1; PP1C, catalytic subunit of PP1; Inh2, protein phosphatase inhibitor-2; GM/GL, glycogen targeting subunit of PP1C; MYPT1, myosin targeting subunit of PP1C; DARPP-32, dopamine and cAMP-regulated phosphoproteins of 32 kDa; CPI-17, protein kinase C-potentiated inhibitor of 17 kDa; PHI-1, phosphatase holoenzymes inhibitor protein; AATYK, apoptosis-associated tyrosine kinase; PP2Ac, catalytic subunit of protein phosphatase 2A; GFP, green fluorescent protein; GST, glutathioneS-transferase; MBP, myelin basic protein; ATPγS, adenosine 5′-[γ-thio]triphosphate; M130, 130-kDa subunit of myosin phosphatase; Nek2, NimA-related kinase; cdk2, cyclin-dependent protein kinase 2; CK-II, casein kinase II; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription; wt, wild type; nt, nucleotide(s)1The abbreviations used are: PP1, protein phosphatase 1; PP1C, catalytic subunit of PP1; Inh2, protein phosphatase inhibitor-2; GM/GL, glycogen targeting subunit of PP1C; MYPT1, myosin targeting subunit of PP1C; DARPP-32, dopamine and cAMP-regulated phosphoproteins of 32 kDa; CPI-17, protein kinase C-potentiated inhibitor of 17 kDa; PHI-1, phosphatase holoenzymes inhibitor protein; AATYK, apoptosis-associated tyrosine kinase; PP2Ac, catalytic subunit of protein phosphatase 2A; GFP, green fluorescent protein; GST, glutathioneS-transferase; MBP, myelin basic protein; ATPγS, adenosine 5′-[γ-thio]triphosphate; M130, 130-kDa subunit of myosin phosphatase; Nek2, NimA-related kinase; cdk2, cyclin-dependent protein kinase 2; CK-II, casein kinase II; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription; wt, wild type; nt, nucleotide(s)is a predominant serine/threonine protein phosphatase that is extraordinarily conserved from yeast to mammalian cells. In the yeastSaccharomyces cerevisiae, PP1C is encoded by GLC7(DIS2) and has over 80% sequence identity with mammalian PP1C (1Feng Z.H. Wilson S.E. Peng Z.Y. Schlender K.K. Reimann E.M. Trumbly R.J. J. Biol. Chem. 1991; 266: 23796-23801Abstract Full Text PDF PubMed Google Scholar). Yeast genetics and mammalian cell biochemistry has shown PP1C is essential for cell survival and multiple cellular functions, including glycogen metabolism, muscle contraction, cell cycle progression, chromosome segregation, and neuronal signaling (2Sakumoto N. Mukai Y. Uchida K. Kouchi T. Kuwajima J. Nakagawa Y. Sugioka S. Yamamoto E. Furuyama T. Mizubuchi H. Ohsugi N. Sakuno T. Kikuchi K. Matsuoka I. Ogawa N. Kaneko Y. Harashima S. Yeast. 1999; 15: 1669-1679Crossref PubMed Scopus (94) Google Scholar, 3Newgard C.B. Brady M.J. O'Doherty R.M. Saltiel A.R. Diabetes. 2000; 49: 1967-1977Crossref PubMed Scopus (149) Google Scholar, 4Ramaswamy N.T. Li L. Khalil M. Cannon J.F. 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These different processes are regulated by distinct PP1 holoenzymes in which the same catalytic subunit interacts with different regulatory subunits (R subunit) (6Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar, 12Aggen J.B. Nairn A.C. Chamberlin R. J. Chem. Biol. 2000; 7: R13-R23Abstract Full Text Full Text PDF Scopus (142) Google Scholar, 13Bollen M. Trends Biochem. Sci. 2001; 26: 426-431Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). R subunits directly restrict activity of PP1C and target the enzyme to a specific subcellular location or substrate. Well known R subunits include the GM/GL subunit, which targets PP1C to glycogen particles (14Tang P.M. Bondor J.A. Swiderek K.M. DePaoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar, 15Hubbard M.J. Dent P. Smythe C. Cohen P. Eur. J. Biochem. 1990; 189: 243-249Crossref PubMed Scopus (62) Google Scholar, 16Doherty M.J. Moorhead G. Morrice N. Cohen P. Cohen P.T. FEBS Lett. 1995; 375: 294-298Crossref PubMed Scopus (140) Google Scholar), and the MYPT1 subunit, which targets PP1C to myosin and moesin/ERM proteins of the actin cytoskeleton (17Chen Y.H. Chen M.X. Alessi D.R. Campbell D.G. Shanahan C. Cohen P. Cohen P.T. FEBS Lett. 1994; 356: 51-55Crossref PubMed Scopus (128) Google Scholar, 18Fukata Y. Kimura K. Oshiro N. Saya H. Matsuura Y. Kaibuchi K. J. Cell Biol. 1998; 141: 409-418Crossref PubMed Scopus (181) Google Scholar). Control of PP1 by R subunits is supplemented by a series of small inhibitor phosphoproteins such as inhibitor-1 (19Endo S. Zhou X. Connor J. Wang B. Shenolikar S. Biochemistry. 1996; 35: 5220-5228Crossref PubMed Scopus (150) Google Scholar), its homologue dopamine and cAMP-regulated phosphoprotein (DARPP-32) (20Kurihara T. Lewis R.M. Eisler J. Greengard P. J. Neurosci. 1988; 8: 508-517Crossref PubMed Google Scholar, 21Huang H.-b. Horichi A. Watanabe T. Shih S.-R. Tsay H.-J. Li H.-C. Greengard P. Nairn A.C. J. Biol. Chem. 1999; 274: 7870-7878Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), CPI-17 (22Eto M. Senba S. Morita F. Yazawa M. FEBS Lett. 1997; 410: 356-360Crossref PubMed Scopus (226) Google Scholar), PHI-1 (23Eto M. Karginov A. Brautigan D.L. Biochemistry. 1999; 38: 16952-16957Crossref PubMed Scopus (89) Google Scholar), and inhibitor-2 (Inh2) (24Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar, 25Yang J. Hurley T.D. DePaoli-Roach A. J. Biol. Chem. 2000; 275: 22635-22644Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Inh2 is common partner of PP1C and is conserved from yeast to human. In the yeast,GLC8 encodes a protein resembling mammalian Inh2, although there is only 28% identity in primary sequence (8Tung H.Y. Wang W. Chan C.S. Mol. Cell. Biol. 1995; 15: 6064-6074Crossref PubMed Scopus (87) Google Scholar, 26Cannon J.F. Pringle J.R. Fiechter A. Khalil M. Genetics J. 1994; 136: 485-503Crossref PubMed Google Scholar). Intracellular localization and isolation of Inh2 and PP1 (27Tung H.Y. Cohen P. Eur. J. Biochem. 1984; 145: 57-64Crossref PubMed Scopus (67) Google Scholar, 28Vandenheede J.R. Agostinis P. Staquet S.I. Van Lint J. Adv. Protein Phosphatases. 1989; 5: 19-36Google Scholar, 29Brautigan D.L. Fernandez A. Lamb N.J.C. Adv. Protein Phosphatases. 1991; 6: 375-390Google Scholar) raised questions as to whether Inh2 is bound to PP1 or might associate with other partners besides PP1. To address these questions, we performed the yeast two-hybrid study using Inh2 as bait. KPI-2 is one of the proteins we identified, cloned, expressed, and characterized. Here we describe the biochemical properties of this novel transmembrane protein, which has a kinase domain and a C-terminal domain that associates with PP1C. KPI-2 is a serine/threonine kinase that can autophosphorylate and also phosphorylate PP1C at Thr320 and inactivate its phosphatase activity. KPI-2 interacts with Inh2 in conjunction with PP1, and this forms a complex where PP1 is associated with two different regulators. Calf thymus Histone H1 and Microcystin-LR were purchased from Calbiochem-Novabiochem. [32P]ATP (30 Ci/mmol) was purchased from Amersham Biosciences. Anti-FLAG M2 antibody and anti-FLAG M2-agarose affinity gel were obtained from Sigma. Mouse anti-PP1C monoclonal antibody was purchased from Transduction Laboratories. Phospho-Thr-Pro monoclonal antibody and phospho-PP1α(Thr320) were from Cell Signaling Technology. Phospho-Ser antibody was from BIOMOL Research Laboratories, Inc. FuGENE 6 transfection reagent was from Roche Molecular Biochemicals. Sf9 cells were obtained from Invitrogen. Restriction enzymes were purchased from New England BioLabs. The library screen was performed by using human Inh2 as bait. Residues 1–197 of Inh2 were inserted into a pGBT10 vector, a derivative of pGBT9 that contains the Gal4 DNA-binding domain. The library was a 9-day embryonic mouse cDNA cloned into pVP16 vector, which contains the Gal4 activation domain (created by Dr. S. Hollenberg, Fred Hutchinson Cancer Center, Seattle, WA). The screening was done by using the large-scale, sequential transformation method (30Zhu L. Meth. Mol. Biol. 1997; 63: 173-196PubMed Google Scholar). 2 × 106 clones were screened. Positive clones were tested first for expression of the HIS3 gene (His+) by growth of the clones on the plates lacking histidine (SD/-Trp/-Leu/-His). The positives of those were tested for expression of the reporter gene, lacZ, using an assay for β-galactosidase activity. Clones were rescued by electroporation intoEscherichia coli HB101 and grown on M9 plates lacking leucine, which allowed for analysis of positives by transformation tests and DNA sequencing. For protein-protein interaction, we used an alternative yeast two-hybrid system. Inh2 or PP1Cα gene was fused to the Gal4 DNA-binding domain in the pGBT10 vector, and KPI-2 C-terminal wild type (residues 1099–1503), AA-mutant, or IB-4 (KPI-2 fragment from two-hybrid screen) was fused to the Gal4-activation domain in a pVP16 vector. Both bait and prey plasmids were cotransformed into HF7c cells. Protein-protein interaction was determined by checking the growth of clones on the plate lacking histidine (SD/-Trp/-Leu/-His). mRNA was isolated from HeLa cells with a QuickPrepTM Micro mRNA purification kit (AmershamBiosciences). N- and C-terminal portions of KPI-2 cDNA were synthesized separately using a One-Step RT-PCR kit (Qiagen). Primers were designed according to the sequence of KIAA1079 cDNA, and the following primers were used (Fig. 1 A): for N-terminal, forward primer containing BamHI site 5′-TAT AATGGATCC ACC ATG CCG GGG CCG CCG GCG TT-3′, reverse primer containing HindIII site 5′-TGT GTT CTT TGC TGG ACA ATG AAGCTT TTA GTA AGT-3′; for C-terminal, forward primer containing HindIII site 5′-ACT TAC TAA AAGCTT CAT TGT CCA GCA AAG AAC ACA-3′, reverse primer containing XhoI site 5′-CACTCGAGG TCC TTT TCT CCG TCT TCG CTG CTT CC-3′. The amplified C terminus was subcloned into a pCMV myc-tagged vector with an HindIII-XhoI site. The full-length KPI-2 was created by inserting the N-terminal portion into the plasmid containing the C-terminal portion at theBamHI-HindIII site. The construct of KPI-2 full-length was named pCMV-KPI-2-full-length. The HindIII site was repaired to original sequence by using a Stratagene QuikChange mutagenesis kit according to the manufacturer's protocol. pCMV-KPI-2-N-terminal was constructed by inserting an N-terminal fragment (1–703 residues, amplified by PCR) into pCMV myc-tagged vector with a BamHI-XhoI site. GST-PKI-2 was constructed by inserting a C-terminal cDNA fragment (amplified by PCR), including PP1C binding motif (residues 1099–1503) into pGEX-Parallel3 vector, and was named pGEX-KPI-2-wt. We also mutated the PP1C binding motif (VTF) of KPI-2 by substituting Val1355 and Phe1357 with Ala to produce pGEX-KPI-2-AA-mut, using the QuikChange mutagenesis kit. FLAG-tagged KPI-2-wt and KPI-2-AA-mut were constructed by ligating aBamHI-XhoI fragment from pGEX-KPI-2-wt or AA-mut into pcDNA3-Flag2AB vector. The fragments of KPI-2 C-terminal wild type-(1099–1503) and AA-mutant were also subcloned into yeast expression vector pVP16; we named these pVP16-KPI-2-wt and pVP16-KPI-2-AA-mut. pFastBac-His6-KPI-2-kinase plasmid was constructed for use in the Bac-to-Bac Baculovirus Expression System (Invitrogen). The KPI-2 fragment, including a kinase domain (residues 94–600), was amplified by PCR and subcloned into pFastBac HTb vector with aBamHI-EcoRI site to form a recombinant pFastBac donor plasmid. All the constructs are shown in Fig. 1 B. All DNA sequences above were confirmed by double-stranded DNA sequencing in the Biomolecular Research Facility of the University of Virginia. Northern blotting was performed using Clontech human Multiple Tissue Northern (MTNTM) Blots according to the manufacturer's instructions (Clontech). The membrane was probed with32P-labeled cDNA corresponding to the kinase domain (280–1800 nt) of KPI-2. The membrane was stripped and reprobed with cDNA encoding the C-terminal domain (3295–4509 nt). After washing, the membrane was exposed to x-ray film with an intensifying screen for 72 h at −70 °C. COS7, HeLa, and HEK293T cells were cultured in Dulbecco's modified Eagle's Medium supplemented with 10% heat-inactivated newborn calf serum (Invitrogen). Cells were grown in 10-cm plates in a humidified incubator at 37 °C and 5% CO2 and subcultured every 2–3 days. Cells were transfected by using FuGENE 6 reagent according to the manufacturer's instructions. After 24 h of transfection, the cells were harvested and lysed with lysis buffer (50 mm Tris/HCl, pH 8.0, 150 mm NaCl, 50 mm NaF, 1% Nonidet P-40, 20 mm β-glycerophosphate, 1 mmNa3VO4, 1 mm dithiothreitol, 0.1% 2-mercaptoethanol, 1 mm Pefabloc-sc, 10 μg/ml leupeptin, and 10 μg/ml pepstatin) for 30 min on ice. The lysates were clarified by centrifugation at 10,000 × g for 10 min. For Western blotting, equal amount of proteins were subjected to SDS-PAGE and immunoblotted with specific antibodies. For immunoprecipitation, the lysates were incubated with anti-FLAG M2-agarose affinity gel for 1 h at 4 °C. The beads were washed three times with the lysis buffer and then subjected to SDS-PAGE and immunoblotted with anti-FLAG, anti-PP1C, or anti-Inh2 antibody. COS7 cells transfected with pCMV-KPI2-N-terminal for 24 h were washed once with phosphate-buffered saline, and scraped in ice-cold buffer containing 10 mm Tris/HCl, pH 8.0, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mmPefabloc-sc, 10 μg/ml leupeptin and 10 μg/ml pepstatin. The cells were disrupted by homogenization, and the homogenates were clarified by centrifugation at 1000 × g for 10 min at 4 °C. The pellet was homogenized again, and the supernatants were pooled and then centrifuged at 4000 × g for 10 min. The supernatants were mixed with an equal volume of the same buffer containing 0.25m sucrose and centrifuged at 100,000 × gfor 1 h. The supernatants were transferred to new tubes, and concentrated by using Centricon Centrifugal Filter Devices (Millipore). The membrane pellets were solubilized with lysis buffer containing 1% Nonidet P-40. The protein concentration was measured, and an equal amount of protein was subjected to SDS-PAGE and immunoblotted with anti-myc antibody. The donor plasmid pFastBac-KPI-2 kinase-(94–600) was transformed into DH10Bac-competent cells for transposition into Bacmid. The recombinant Bacmid DNA was identified by PCR and transfected into Sf9 cells and incubated for 5 days at 27 °C. The recombinant baculoviruses were harvested to obtain the P1 viruses and then amplified with P1 viruses to produce high titer P2, P3 viruses. The virus titer was measured by the cell culture center of the University of Virginia. For protein expression, Sf9 cells were infected with recombinant baculoviruses, and cells were collected 60 h later by centrifugation. The cell pellet was resuspended in lysis buffer (20 mm imidazole-HCl, pH 7.0, 20 mm potassium phosphate, 150 mm NaCl, 1% Nonidet P-40, 0.1% β-mercaptoethanol, 1 mm dithiothreitol, and proteinase inhibitors as above), snap-frozen in liquid nitrogen, then subsequently thawed on ice. The samples were sonicated to lyse all the cells. After centrifugation, the supernatants were incubated with nickel-nitrilotriacetic acid resin for 1 h at 4 °C and transferred to a column. The column was washed with buffer containing 20 mm imidazole-HCl, pH 7.0, 20 mm potassium phosphate, 300 mm NaCl, 10% glycerol, 0.1% β-mercaptoethanol, 1 mm dithiothreitol, and proteinase inhibitors. The proteins were eluted with 150 mm imidazole, and fractions of 0.5 ml were collected. An aliquot of 10 μl from each fraction was subjected to SDS-PAGE and analyzed by Coomassie Blue staining and anti-His immunoblotting. The fractions containing recombinant kinase were pooled and were further purified by Mono Q anion-exchange chromatography using an NaCl gradient from 100 to 600 mm. A lysate from Sf9 cells transfected with wild type baculovirus was processed in parallel. Autophosphorylation was performed by incubating purified recombinant KPI-2 kinase at 30 °C for 20 min in 20 μl of reaction buffer containing 20 mm Hepes, pH 7.4, 1 mm MnCl2, 10 mmMgCl2, 5 mm β-glycerolphosphate, 0.1 mm Na3VO4, 2 mmdithiothreitol, 0.4 mm Pefabloc-sc, and 100 μm [32P]ATP (10 μCi). The reaction was terminated by the addition of 6× SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE, and the gel was stained with Coomassie Blue. The phosphorylation was detected by autoradiography and quantitated by excising the band corresponding to the protein and measuring the radioactivity with a scintillation counter. The substrate phosphorylation used 0.25 mg/ml myelin basic protein (MBP), Histone H1, recombinant His6-Inh2, or poly(Glu:Tyr) (4:1) under the same conditions. The eluant from Sf9 cells transfected with wild type baculovirus was used as negative control for all the kinase assays. Following kinase reaction and autoradiography, the band corresponding to the KPI-2 kinase and MBP were excised. The samples were digested by trypsin overnight followed by acid hydrolysis for 1 h in 6 m HCl at 100 °C. Phosphoamino acid analysis was carried out using one-dimensional thin layer electrophoresis at pH 2.5 (31Jelinek T. Weber M. BioTechniques. 1993; 15: 629-630Google Scholar). The phosphoamino acids were detected by autoradiography. Phosphoamino acid standards were included in the samples, and their locations were determined by Ninhydrin staining. Both GST-KPI-2-wt and GST-KPI-2-AA-mut were expressed in E. coli DH5α and purified with glutathione-Sepharose beads. The GST-KPI2-wt or AA-mut fusion protein (2 μg) was bound to glutathione-Sepharose beads and incubated with HeLa cell lysates at 4 °C for 1 h as described previously (32Wu J. Kleiner U. Brautigan D.L. Biochemistry. 1996; 35: 13858-13864Crossref PubMed Scopus (30) Google Scholar). The beads were washed extensively, and PP1C binding was detected by immunoblotting with anti-PP1C antibody. The content of the GST fusion protein was determined by immunoblotting with anti-GST antibody. PP1C activity was assayed by the release of 32P phosphate from32P-labeled phosphorylase A as described in Shenolikar and Ingebritsen (33Shenolikar S. Ingebritsen T.S. Methods Enzymol. 1984; 107: 102-129Crossref PubMed Scopus (75) Google Scholar). Activity of PP1C purified from rabbit skeletal muscle was determined in a reaction mixture (40 μl) containing 20 mm MOPS, pH 7.4, 2 mmMgCl2, 1 mg/ml bovine serum albumin, and 15 μm32P-labeled phosphorylase A at 30 °C for 15 min. Acid-soluble 32P was analyzed by liquid scintillation counting. To identify binding proteins for Inh2, we employed yeast two-hybrid analysis with human Inh2 (residues 1–197) as bait. One of six independent clones was a fragment of an uncharacterized protein encoded by the human open reading frame of KIAA1079 (NM-011575). The fragment identified in the Inh2 screen, called IB-4, was the region of residues 1344–1450, including the PP1C binding motif VTF (Fig.1 A). We cloned the cDNA from HeLa cells by using RT-PCR and named it KPI-2 (Kinase/Phosphatase/Inhibitor-2). Our KPI-2 cDNA clone was 4512 nucleotides in length encoding a 1503-amino acid residue protein (GenBankTM accession numberAY130988) and matched with human chromosome 7q21.3-q22.1. The KIAA1079 sequence differs from KPI-2 by missing 94 nucleotides that were thought to be an intron with a mismatched splicing junction. The domain structure of KPI-2 protein is shown in Fig. 1 A. There are two predicted transmembrane helices (available at www.enzim.hu/hmmtop/) that extend between amino acid residue ranges 11–29 and 46–63 near the N terminus. There is one kinase domain (residues 137–407) (www.kinase.com), which contains an ATP binding motif (residues 143–168) and has about 60% sequence identity with mouse apoptosis-associated tyrosine kinase (AATYK) (34Gaozza E. Baker S.J. Vora R.K. Reddy E.P. Oncogene. 1997; 15: 3127-3135Crossref PubMed Scopus (65) Google Scholar, 35Raghunath M. Patti R. Bannerman P. Lee C. Baker S. Sutton L. Phil P. Damodar R. Mol. Brain Res. 2000; 5: 151-162Crossref Scopus (59) Google Scholar, 36Baker S.J. Sumerson R. Reddy C.D. Berrebi A.S. Flynn D.C. Reddy E.P. Oncogene. 2001; 20: 1015-1021Crossref PubMed Scopus (27) Google Scholar, 37Tomomura M. Fernandez-Gonzales A. Yano R. Yuzaki M. Oncogene. 2001; 20: 1022-1032Crossref PubMed Scopus (35) Google Scholar). In the C-terminal region, there is a predicted PP1C binding motif KKAVTFFD that contains key Val and Phe at amino acid residue 1355 and 1357. This site was recovered in the two-hybrid clone IB-4. To examine the expression of KPI-2 in different tissues, we probed a Clontech Human Multiple Tissue Northern (MTNTM) blot with a KPI-2 kinase domain (280–1800 nt). KPI-2 mRNA was detected as a single band at size ∼10 kb, which was expressed predominantly in skeletal muscle, with low level expression in brain and pancreas (Fig.1 C). The result was confirmed by reprobing the same membrane with a cDNA corresponding to the KPI-2 C-terminal domain (3295–4509 nt, not shown). Because KPI-2 is expressed mostly in skeletal muscle, we checked by RT-PCR the expression of KPI-2 in C2C12 cells, a mouse myoblast cell line. Consistent with the Northern blot, KPI-2 was expressed in C2C12 cells, but its expression level did not change before and after induction of muscle differentiation (not shown). The full-length KPI-2 protein and its N-terminal (1–703 residues) were expressed in HEK293T cells and detected by Western blotting with anti-myc antibody. Recombinant KPI-2 protein was ∼210 kDa, and the N-terminal-(1–703) was an ∼90-kDa protein as expected from their sequences (Fig.2 A). Because of the predicted transmembrane helices in the N terminus, we expected the KPI-2 protein to be membrane-bound. To test this, we expressed KPI-2 N-terminal-(1–703) in COS7 cells, and prepared soluble and membrane fractions from these cells. The myc-tagged KPI-2-(1–703) protein was recovered entirely in the membrane fraction solubilized by 1% Nonidet P-40 and did not appear in the soluble fraction (Fig. 2 B). Phospholemman was used as a plasma membrane protein marker, and protein phosphatase 2A catalytic subunit was used as cytosolic protein marker. The results show that KPI-2 N-terminal-(1–703) is a membrane protein. Fusion of KPI-2 residues 7–139 to green fluorescent protein (GFP) also resulted in predominant distribution of fusion protein into membrane fraction (not shown). This suggests that the predicted transmembrane helices are sufficient for targeting KPI-2 or fusion proteins to membranes. We expressed the KPI-2 kinase domain (residues 94–600) as a His6-tagged fusion protein in Sf9 cells. Following metal-ion affinity chromatography, one major protein of ∼75 kDa was detected in the Coomassie Blue-stained gel (Fig.3 A, left panel), and this protein also reacted with anti-His antibody (Fig.3 A, right panel). The 75-kDa KPI-2 kinase from Sf9 cells was purified by Mono Q anion-exchange chromatography (not shown), and an autophosphorylation assay was performed. The purified recombinant kinase was incubated with [32P]ATP, and autophosphorylation was observed (Fig. 3 B, left panel). We also performed a kinase assay using different substrates. Myelin basic protein (MBP) was phosphorylated efficiently, but Histone H1 and His6-Inh2 were relatively poor substrates under the same conditions (Fig. 3 B, right panel). The tyrosine kinase substrate poly(Glu:Tyr) (4:1) was not phosphorylated at all, even with prolonged incubation (data not shown). A lysate from Sf9 cells transfected with wild type baculovirus was processed in parallel, and the corresponding Mono Q eluant was used as negative control for all the kinase assays. These results showed that purified KPI-2 protein is an active kinase. We next determined the residues phosphorylated by KPI-2 kinase. After autoradiography, the bands corresponding to the autophosphorylated KPI-2 kinase and the phosphorylated substrate MBP were excised, and phosphoamino acid analysis was carried out using one-dimensional thin layer electrophoresis. The results showed that both KPI-2 autophosphorylation and MBP phosphorylation were mostly located at serine, with a trace at threonine. No tyrosine phosphorylation was observed (Fig. 3 C). Immunoblotting with anti-phospho-Thr-Pro and anti-phospho-Ser antibody also showed reactivity with autophosphorylated KPI-2. This evidence shows that KPI-2 is a serine/threonine protein kinase. KPI-2 is a serine/threonine protein kinase with a PP1 binding motif in the C-terminal domain that would bring the kinase and phosphatase together. We checked whether KPI-2 could phosphorylate PP1C and alter its activity. Purified PP1C was incubated with or without purified KPI-2 kinase-(94–600). Reactions contained 100 μm ATPγS, which was used instead of ATP to retard dephosphorylation of PP1C during the reaction. Phosphatase assays were performed using [32P]phosphorylase a as substrate. PP1C activity was reduced 70% by reaction with KPI-2 kinase compared with incubation without kinase (Fig.4 A). PP1C is known to be inactivated by phosphorylation at Thr320 (38Dohadwala M. da Cruz Hall F.L. Williams R.T. Carbonaro-Hall D.A. Nairn A.C. Greengard P. Berndt N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6408-6412Crossref PubMed Scopus (224) Google Scholar, 39Kwon Y.G. Lee S.Y. Choi Y. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2168-2173Crossref PubMed Scopus (177) Google Scholar, 40Berndt N. Dohadwala M. Liu C.W. Cur. Biol. 1997; 7: 375-386Abstract Full Text Full Text PDF PubMed Google Scholar), so we tested if the phosphorylation by KPI-2 was located at the Thr320 site in PP1C. PP1C was inhibited by incubation with 1 μm microcystin-LR and incubated with ATP plus/minus KPI-2 kinase. Western blotting using anti-phospho-PP1Cα(Thr320) antibody showed that PP1C was phosphorylated at Thr320 by KPI-2 kinase (Fig.4 B). Thiophosphorylated PP1C did not react with the phospho-specific antibody. Thus, KPI-2 phosphorylated Thr320 in PP1C, which reduces phosphatase activity. There is a consensus PP1C binding motif VTF in the C-terminal domain of KPI-2. To check PP1C binding, we prepared GST-KPI-2-(1099–1503) and used HeLa cell lysates as a source of PP1C in a pull-down assay. Wild type (wt) GST-KPI-2-(1099–1503) bound PP1C, but GST alone as a" @default.
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• W2087422952 title "A Novel Transmembrane Ser/Thr Kinase Complexes with Protein Phosphatase-1 and Inhibitor-2" @default.
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