Functional Interaction between the Ser/Thr Kinase PKL12 and N-Acetylglucosamine Kinase, a Prominent Enzyme Implicated in the Salvage Pathway for GlcNAc Recycling
2002; Elsevier BV; Volume: 277; Issue: 8 Linguagem: Inglês
10.1074/jbc.m105766200
ISSN1083-351X
AutoresJosé M. Ligos, Teresa Laı́n de Lera, Stephan Hinderlich, Bárbara Guinea, Luis Sánchez‐Pulido, Ramón Roca, Alfonso Valencia, António Bernad,
Tópico(s)Protein Tyrosine Phosphatases
ResumoPKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control. Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. One positive clone contained an open reading frame highly similar toN-acetylglucosamine kinase (GlcNAcK) of several species. The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo. Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues. Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs. After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein. GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo. In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein. These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins. PKL12 (STK16) is a ubiquitously expressed Ser/Thr kinase, not structurally related to the well known subfamilies, with a putative role in cell adhesion control. Yeast two-hybrid protein interaction screening was used to search for proteins that associate with PKL12 and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. One positive clone contained an open reading frame highly similar toN-acetylglucosamine kinase (GlcNAcK) of several species. The PKL12/GlcNAcK interaction was further confirmed both in vitro and in vivo. Protein expression analysis of GlcNAcK using a specific rabbit antiserum displayed a ubiquitous pattern in cell lines and animal tissues. Subcellular localization studies showed that GlcNAcK is a cytoplasmic protein with a dual subcellular localization, distributed between the perinuclear and peripheral cell reservoirs. After overexpression, GlcNAcK localizes in vesicular structures associated mainly with the cell membrane and colocalizes with the PKL12 protein. GlcNAcK is not otherwise a substrate for PKL12 activity and PKL12 does not appear to influence GlcNAcK activity either in vitro or in vivo. In vitro kinase assays have nonetheless revealed that functional GlcNAcK, although not able to modulate autophosphorylation of PKL12, greatly influences PKL12 kinase activity on a defined substrate protein. These results are interpreted to indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins. PKL12 (protein kinase expressed in day 12 fetal liver; also known as kinase related to Saccharomyces cerevisiae andArabidopsis thaliana (Krct), embryo-derived protein kinase (EDPK), and myristoylated and palmitoylated serine-threonine kinase-1 (MPSK1)) has recently been isolated from several sources and partially characterized (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar, 2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar, 3Kurioka K. Nakagawa K. Denda K. Miyazawa K. Kitamura N. Biochim. Biophys. Acta. 1998; 1443: 275-284Crossref PubMed Scopus (16) Google Scholar, 4Berson A.E. Young C. Morrison S.L. Fuji G.H. Sheung J., Wu, B. Bolen J.B. Burkhard A.L. Biochem. Biophys. Res. Commun. 1999; 259: 533-538Crossref PubMed Scopus (20) Google Scholar). All correspond to the same mammalian gene, human or murine, for which the denominationSTK16 has been proposed (International Committee for Human Nomenclature). PKL12 protein appears to be the first mammalian member of a new Ser/Thr kinase subfamily not closely related to those reported previously (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar). This subfamily includes the putative homologues from S. cerevisiae and A. thaliana, forming a group of four sequences close in size to a theoretical minimal catalytic domain. It has therefore been proposed that PKL12 may be the catalytic subunit of a more complex holoenzyme composed of catalytic and regulatory subunits (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar). Escherichia coli-expressed PKL12, His-tagged or as a GST 1GSTglutathioneS-transferaseGlcNAcKN-acetylglucosamine kinasehhumanmmurinePBSphosphate-buffered salineHAhemagglutinin 1GSTglutathioneS-transferaseGlcNAcKN-acetylglucosamine kinasehhumanmmurinePBSphosphate-buffered salineHAhemagglutinin fusion, and a FLAG-tagged PKL12 protein have been shown to have functional kinase activity, able to phosphorylate exogenous substrates (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar, 2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar, 3Kurioka K. Nakagawa K. Denda K. Miyazawa K. Kitamura N. Biochim. Biophys. Acta. 1998; 1443: 275-284Crossref PubMed Scopus (16) Google Scholar) and to promote autophosphorylation (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar, 2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar) with the sequence-predicted Ser/Thr specificity (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar, 3Kurioka K. Nakagawa K. Denda K. Miyazawa K. Kitamura N. Biochim. Biophys. Acta. 1998; 1443: 275-284Crossref PubMed Scopus (16) Google Scholar). glutathioneS-transferase N-acetylglucosamine kinase human murine phosphate-buffered saline hemagglutinin glutathioneS-transferase N-acetylglucosamine kinase human murine phosphate-buffered saline hemagglutinin PKL12 mRNA appears to be broadly distributed, both in murine fetal stages (E6.5 to E18.5) (2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar, 3Kurioka K. Nakagawa K. Denda K. Miyazawa K. Kitamura N. Biochim. Biophys. Acta. 1998; 1443: 275-284Crossref PubMed Scopus (16) Google Scholar) and in adult tissues, at low levels in skeletal muscle, heart, and spleen, and with high expression in liver, testis, and kidney (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar, 2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar, 3Kurioka K. Nakagawa K. Denda K. Miyazawa K. Kitamura N. Biochim. Biophys. Acta. 1998; 1443: 275-284Crossref PubMed Scopus (16) Google Scholar). Despite its ubiquitous distribution,in situ analysis showed that PKL12 mRNA is preferentially expressed within specific cellular types in several adult tissues, with predominant expression in epithelial compared with mesenchymal compartments (2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar). Further analyses have confirmed the broad distribution of the PKL12 protein in murine tissues and cell lines, although a lack of correlation between mRNA and protein levels was reported, 2J. M. Ligos, M. T. Laı́n de Lera, B. Guinea, J. Martı́n-Caballero, J. Flores, and A. Bernad, submitted for publication. 2J. M. Ligos, M. T. Laı́n de Lera, B. Guinea, J. Martı́n-Caballero, J. Flores, and A. Bernad, submitted for publication. suggesting post-transcriptional regulation (2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar). hPKL12 is acylated by myristic acid at glycine residue 2 and by palmitic acid at cysteines 6 and/or 8 (4Berson A.E. Young C. Morrison S.L. Fuji G.H. Sheung J., Wu, B. Bolen J.B. Burkhard A.L. Biochem. Biophys. Res. Commun. 1999; 259: 533-538Crossref PubMed Scopus (20) Google Scholar). It has been proposed that hPKL12 membrane localization via a myristoylation-dependent mechanism is required for the subsequent palmitoylation modification, as demonstrated in other models (4Berson A.E. Young C. Morrison S.L. Fuji G.H. Sheung J., Wu, B. Bolen J.B. Burkhard A.L. Biochem. Biophys. Res. Commun. 1999; 259: 533-538Crossref PubMed Scopus (20) Google Scholar, 5Berthiaume L. Resh M.D. J. Biol. Chem. 1995; 270: 22399-22405Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). A membrane-associated protein kinase, PKL12, must therefore play a role in intracellular signaling, with a general, highly conserved cellular function.2Subcellular localization analysis has shown that PKL12 is a Golgi-resident enzyme. Transient overexpression of PKL12 in NIH-3T3 cells promotes its accumulation in structures related to filopodia and lamellipodia, inducing redistribution of focal contacts and disorganization of the actin cytoskeleton but no marked alterations in Golgi.2 A regulatory role is thus hypothesized for PKL12 in the control of extracellular matrix-cell adhesion, mediating the dynamic equilibrium of organization/disorganization of focal adhesion structures and actin cytoskeleton.2 Concurring with this proposal, high level forced expression of PKL12 protein in adherent cell lines appears to be incompatible with their survival, whereas PKL12 can be overexpressed in non-adhesion-dependent cell lines without disturbing growth and survival parameters.2 Based on a two-hybrid analysis, we have identified and demonstrated functional interaction in vitro and in vivobetween PKL12, a Golgi-resident Ser/Thr kinase, and a recently cloned enzyme (6Hinderlich S. Berger M. Schwarzkop M. Effertz K. Reutter W. Eur. J. Biochem. 2000; 267: 3301-3308Crossref PubMed Scopus (66) Google Scholar) of amino sugar metabolism, N-acetylglucosamine kinase (GlcNAcK). Although GlcNAcK is not a substrate of PKL12, nor does PKL12 influence GlcNAcK activity either in vitro orin vivo, we have found that both enzymes colocalize in vivo upon overexpression, being a functional GlcNAcK capable of influencing PKL12 kinase activity on exogenous substrates. These results indicate a potential in vivo role for GlcNAcK in PKL12 translocation and a tentative regulatory role for PKL12-mediated phosphorylation on substrate proteins. NIH-3T3 and SV40-transformed NIH-3T3 cells were obtained from the American Type Culture Collection (Manassas, VA); BA/F3, A.20, FL5.12, WEHI3b, ST2, and EL4 cells were kindly provided by Dr. C. Martı́nez-A (Centro Nacional de Biotecnologia, Madrid, Spain); COS-1 cells were a kind gift of Dr. J. Ortı́n (Centro Nacional de Biotecnologia, Madrid, Spain). NIH-3T3, COS-1, and ST2 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mml-glutamine (Merck), streptomycin (0.1 mg/ml, Sigma), and penicillin (100 units/ml, Sigma). A.20, BA/F3, FL5.12, WEHI3b, and EL4 cells were maintained in RPMI 1640 (Invitrogen) supplemented as above. BA/F3 stable clones (K3 and K8) overexpressing PKL12 protein have been described.2 The yeast two-hybrid protein interaction screen was carried out essentially as described (7McNabb D.S. Guarantee L. Curr. Opin. Biotechnol. 1996; 7: 554-559Crossref PubMed Scopus (14) Google Scholar) using the HF7c yeast strain (CLONTECH, Palo Alto, CA). Murine PKL12 was fused to the GAL4 DNA-binding domain in the pGBT8 vector (kindly provided by Dr. M. Serrano), generating the pGBT8-PKL12 vector. The coding sequence for the murine PKL12 orf was amplified by PCR from the pcDNA3-PKL12 plasmid, using specific primers (sense primer, 5′-GATGTCGAATTCTTAATGGGCCACGCACTG-3′; antisense primer, 5′-CGACTGAGATCTTCAGATTGGGTGGTGTG-3′) for simultaneous elimination of the PKL12 orf initiation codon and creation of an EcoRI site for in-frame fusion with the GAL4 DNA binding domain in pGBT8. PCR conditions were as follows: 94 °C, 30 s; 60 °C, 1 min; 68 °C, 10 min for 12 cycles, using theTaq/Pow thermostable enzyme mixture (Expand Long Template System; Roche Molecular Biochemicals) and a 2400 PerkinElmer Life Sciences Thermocycler. The amplified products were digested withEcoRI and BglII and ligated to the pre-digested vector and transformed in E. coli XL1-blue competent cells. An NIH-3T3 cDNA library generated in the pGAD424 vector (provided by Dr. M. Serrano) was used in the screening. Yeasts stably transformed with the pGBT8-PKL12 vector, and thus able to grow in tryptophan-free medium, were obtained. Stable expression of GAL4bd-mPKL12 protein was confirmed by Western blot (not shown). Yeasts were then transformed with the NIH-3T3 cDNA library in the pGAD424 vector and selected for growth in minimal medium without tryptophan, leucine, or histidine. As positive controls, pGBT8-p16 and pGAD424-cdk4 plasmids were cotransfected in yeast; as negative controls the pGBT8 vector plasmid was cotransfected with the pGAD424-cdk4 plasmid. Yeast clones obtained after selection in tryptophan-, leucine-, and histidine-free medium were also tested for β-galactosidase expression. After isolation of pGAD424-derivative plasmids from clones positive for both selective criteria, they were re-confirmed by direct cotransfection with the pGBT8-PKL12 vector in HF7c yeast, and inserts were directly sequenced using the vector primers. Specific oligonucleotides (sense primer, 5′-AGGCGACACAGGGGCGAGAGA-3′; antisense primer, 5′-GAAAGCGGTGCCTCAACTCCTC-3′) were synthesized (Isogen, Maarssen, The Netherlands) based on the 5′ and 3′ sequences of the mSIP16 obtained from the ESTs data bank and our data. Total mRNA was prepared from NIH-3T3 cells and used to obtain cDNA. PCR was carried out on cDNA derived from NIH-3T3 cells with the primers indicated above (94 °C, 1 min; 60 °C, 1 min; 72 °C, 1,5 min; 30 cycles), using Taq/Pow as above. The fragment obtained, of the predicted size, was cloned in the pGEM-T (Promega) plasmid. Several clones were obtained and fully sequenced. The final clone selected was termed pGEM-GlcNAcK. The pcDNA3-PKL12 plasmid was obtained by subcloning mPKL12 into the pcDNA3 eukaryotic expression plasmid (Invitrogen, Carlsbad, CA) as described.2pcDNA3-HA GlcNAcK plasmid was obtained by subcloning the GlcNAcKorf isolated from the pGEM-GlcNAcK plasmid. Specific oligonucleotides were synthesized (sense primer, 5′-TACTGAGATCTTTGGCCGCGCTTTATGGTGG; antisense primer, 5′-GAAAGCGGTGCCTCAACTCCTC-3′) to create an EcoRI site for in-frame SIP16 orf fusion with the hemagglutinin epitope (HA) in the pcDNA3.1/Neo-HA plasmid. PCR conditions were as follows: 94 °C, 30 s; 60 °C, 1 min; 68 °C, 10 min for 12 cycles. The amplified products were digested with EcoRI, ligated to the EcoRI-digested vector, and transformed inE. coli XL1-blue competent cells. Several clones were obtained, and their sequences were completely confirmed. All plasmids were purified using the Plasmid Maxi Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Histidine-tagged PKL12 was expressed in E. coli strain M15 and purified to homogeneity using nickel-nitrilotriacetic acid-agarose resin (Qiagen), as described (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar); GST-SIP16 protein expression and purification was performed in a similar manner. The coding sequence for the orfcorresponding to murine SIP16 was PCR-amplified from the pGAD424-SIP16 plasmid using specific primers (sense primer, 5′-TACCTGAGATCTACAGACCAGTGTGTGG-3′; antisense primer, 5′-ATACGAATTCACTAGAAGGTATAGGAATA-3′) for simultaneous elimination of the SIP16 open reading frame initiation codon and creation of aBamHI site for in-frame fusion with the GST protein in the pGEX-2T plasmid (Amersham Biosciences). PCR conditions were as follows: 94 °C, 30 s; 60 °C, 1 min; 68 °C, 10 min for 12 cycles. Amplified products were BamHI-digested, ligated to the digested vector, and transformed in E. coli XL1-blue competent cells. Several clones were obtained and their sequences completely confirmed. GST-tagged SIP16 protein was expressed inE. coli (XL1B strain) and purified using the GST Gene Fusion System (Amersham Biosciences) following manufacturer's guidelines.E. coli extracts were prepared as described (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar); those containing the expressed GST-SIP16 protein were loaded in a prepacked glutathione-Sepharose 4B column (Amersham Biosciences). After extensive washing, retained protein was eluted with buffer E (50 mmTris-HCl, pH 8.0, 10 mm glutathione). Purification was monitored by SDS-PAGE (8Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar) of aliquots of the fractions obtained, followed by Coomassie Blue staining or Western blot analysis with an anti-GST antibody (Santa Cruz, Santa Cruz, CA). A parallel negative control purification was performed using E. coli cells transformed with the vector pGEX-2T plasmid. Several independent induction/purification experiments rendered an almost pure fraction of GST-SIP16 protein, with two protein bands (see under “Results,” Fig. 3). Both bands were recognized by anti-GST antibody and the specific anti-SIP16 antiserum; we thus concluded that in E. coli or during the purification process, the SIP16 protein is proteolyzed at a specific carboxyl-terminal point, rendering both products. These bands are not present in negative control purified fractions. Control GST and GST-SP (a fusion of GST with a protein that is a substrate for the kinase activity of the PKL12 protein)2proteins were expressed and purified essentially as described for GST-SIP16. Protein concentration of purified fractions was determined using the Bio-Rad protein assay with a bovine serum albumin standard. Purified GST-tagged SIP16 protein was prepared as above. Outbred New Zealand rabbits were injected intradermally in multiple sites using 250 μg of purified protein emulsified with an equal volume of Freund's complete adjuvant. Two 125-μg intramuscular boosts of the same material in incomplete adjuvant were given 4 and 7 weeks later. Sera were collected 7 and 10 days after the last injection and tested in enzyme-linked immunosorbent assay; the IgG serum fraction was purified as described (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar). Cells (2 × 106) were transfected by electroporation (200 V, 960 microfarads in 0.2 ml) in a Gene Pulser (Bio-Rad) with 20 μg of pcDNA3-PKL12 or pcDNA3-HA-GlcNAcK plasmids and then cultured in standard conditions or on coverslips. After 18 h, cells were processed for Western blot analysis or fluorescence microscopy. Cells (2 × 106) were lysed in 100 μl of RIPA buffer (137 mm NaCl, 20 mm Tris-HCl, pH 8.0, 1 mm MgCl2, 1 mm CaCl2, 10% glycerol, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mmphenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin) for 30 min at 4 °C, and cellular debris was removed by centrifugation (18,000 × g, 20 min). Protein content was determined using the Bio-Rad protein assay (Bio-Rad). Protein (20 μg) was separated in SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked for 1 h in Tris-buffered saline (25 mm Tris; TBS) plus 0.1% Tween, with 5% non-fat dry milk, followed by incubation with primary antibody for 2 h and secondary antibody for 40 min. Western blots were developed using the ECL system (Amersham Biosciences). The polyclonal anti-PKL12 and anti-GlcNAcK antisera were used at 1:2000 and 1:3000 dilution, respectively. Cells were transfected as above, cultured for an additional 24 h, and lysed in 100 μl of IP buffer (150 mm NaCl, 100 mm Tris-HCl, pH 7.4, 10% glycerol, 1% Nonidet P-40, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin) for 30 min at 4 °C, and cellular debris was removed by centrifugation. Protein content was determined as above. Protein (200 μg) in 0.5 ml of IP buffer was incubated with 5 μl of polyclonal anti-PKL12 antibody or 5 μl of polyclonal anti-GlcNAcK antibody (16 h, 4 °C), followed by incubation with protein A-Sepharose beads (Sigma) for 1 h. After extensive washing with IP buffer, immune complexes were analyzed by SDS-PAGE and Western blotting. Cells were fixed in 4% paraformaldehyde (10 min, room temperature) and then washed three times in PBST (PBS containing 0.1% Tween). Preparations were incubated with PBS plus 20% fetal calf serum (1 h, room temperature) followed by primary antibody (1 h). After washing with PBST and staining with secondary antibody (40 min), cells were washed and mounted in ProLong Antifade mounting medium (Molecular Probes, Eugene, OR). Polyclonal anti-PKL12 and anti-GlcNAcK antisera were used at 1:150 and 1:100 dilutions, respectively. Murine anti-HA monoclonal antibody (Roche Molecular Biochemicals) was used at 1:1000 dilution. Secondary antibodies Alexa 488-conjugated goat anti-rabbit IgG, Cy3-goat anti-rabbit IgG, and Cy2-goat anti-mouse IgG (Jackson ImmunoResearch) were used following the manufacturer's instructions. In vitro kinase assays using purified proteins were performed as follows: 35 μl of reaction buffer containing the indicated amount of recombinant histidine-tagged PKL12, 50 mm Tris-HCl, pH 7.4, 10 mm MnCl2, 10 mm MgCl2, 10 mm ATP, and 10 μCi/μl of [γ-32P]ATP, 3000 Ci/mmol (Amersham Biosciences), were preincubated (30 °C, 1 min) and then mixed with 10 ml of the same buffer containing the indicated amounts of the purified putative substrate and/or modulator proteins. Reactions were incubated (30 °C, 30 min), terminated by addition of Laemmli sample buffer, and proteins separated in 10% SDS-PAGE gels. After Coomassie Blue staining, the gel was dried and autoradiographed. As a negative control, equivalent fractions purified from E. coli vector-transformed clones were assayed. BAF/3 PKL12-stable clones cells (2 × 106) or transiently transfected NIH-3T3 or COS-1 cells (2 × 106) were prepared as described.2PBS-washed cells were lysed by hypotonic shock in 200 μl of 10 mm sodium phosphate, pH 7.5, 1 mmdithiothreitol, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride. The lysate was centrifuged (30,000 × g, 20 min), the supernatant assayed for GlcNAcK activity, and analyzed for GlcNAcK expression in Western blot. In vitro GlcNAcK activity was determined as described (7McNabb D.S. Guarantee L. Curr. Opin. Biotechnol. 1996; 7: 554-559Crossref PubMed Scopus (14) Google Scholar). In brief, GlcNAcK assays were performed in a final volume of 225 μl containing 60 mm Tris-HCl, pH 7.5, 20 mmMgCl2, 5 mm GlcNAc, 10 mm ATP (disodium salt), 10 mm phosphoenolpyruvate, 2.5 units of pyruvate kinase, 50 nCi of [1-14C]GlcNAc, and variable amounts of protein extract. Incubations were carried out (37 °C, 2 h), and the reaction was terminated by adding 350 μl of ethanol. Radiolabeled substrates were separated by descendent paper chromatography and measured by liquid scintillation analysis. Initial comparative sequence searches were performed using the BLAST algorithm (9Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69694) Google Scholar) on a non-redundant data base (EMBL NRDB). Additional remote homologue searching was done by HMM (hmm search, default parameters) over protein NRDB data base (10Durbin R. Eddy S. Krogh A. Mitchison G. The Theory Behind Profile HMMs: Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids. Cambridge University Press, Cambridge, UK1998Google Scholar). Sequences were aligned with ClustalW software (11Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 24: 4876-4882Crossref Scopus (35296) Google Scholar). Alignment visualization was performed with Belvu (E. Sonnhammer, www.cgr.ki.se/cgr/groups/sonnhammer/Belvu.html). Complementary data and alignments are available on request at www.cnb.uam.es/∼cnbprot/priv/STK/SIP16. We used the yeast two-hybrid protein interaction screen to search for proteins able to associate with the murine PKL12 (STK16) Ser/Thr kinase and to delineate signaling pathways and/or regulatory circuits in which this kinase participates. PKL12 is expressed ubiquitously, and NIH-3T3 cells are a study model (1Ligos J.M. Gerwin N. Fernandez P. Gutierrez-Ramos J.C. Bernad A. Biochem. Biophys. Res. Commun. 1998; 249: 380-384Crossref PubMed Scopus (18) Google Scholar, 2Stairs D.B. Perry Gardner H., Ha, S.I. Copeland N.G. Gilbert D.J. Jenkins N.A. Chodosh L.A. Hum. Mol. Genet. 1998; 7: 2157-2166Crossref PubMed Scopus (22) Google Scholar, 3Kurioka K. Nakagawa K. Denda K. Miyazawa K. Kitamura N. Biochim. Biophys. Acta. 1998; 1443: 275-284Crossref PubMed Scopus (16) Google Scholar).2 HF7c yeast was stably transformed with the GAL4bd-PKL12 plasmid (pGBT8-PKL12); a NIH-3T3 library fused to the GAL4 activator domain in the pGAD424 plasmid was then transformed and screened in minimal medium. Several clones were selected that grew under the selection conditions and also expressed lacZ, the second transactivable marker gene. pGAD424 plasmids harbored by the positive yeast clones were isolated; positive interaction was confirmed by individual cotransformation of HF7c yeast with pGBT8-PKL12 plasmid or the negative controls pGBT8-p16 (12Serrano M. Hannon G.J. Beach D. Nature. 1993; 336: 704-707Crossref Scopus (3364) Google Scholar) and pGBT8. Positive clones were isolated and fully sequenced. Several positive clones (representing 23% of sequences obtained) were found containing partial sequences corresponding to the same gene. The clone containing the longest open reading frame corresponded to a 334-amino acid protein (38 kDa) in phase with GAL4db. This clone, pGAD424-SIP16Δ, was selected for some of the additional experiments. This protein was denominated SIP16 (STK-16 interacting protein), as sequence homology analysis showed that it has a yet undescribed function. Homologous EST sequences from several murine and human tissues and a Caenorhabditis elegans (WO6B4.2) protein (unknown function) showed significant similarity to the SIP16Δ encoded orf. Based on the data bank-annotated EST sequences, we identified the putative 5′-untranslated region sequence and the transcription initiation codon of the mSIP16 orf. We designed specific oligonucleotides to clone the full-length cDNA, which was obtained from NIH-3T3 cells. mSIP16 protein consists of 343 amino acids (approximate mass 40 kDa), and hSIP16 was electronically assembled from the data bank EST sequences. Similarity searches using human and murine sequences (92% similarity) revealed the presence of ortholog sequences in several other eukaryotic organisms (Fig. 1), includingDrosophila melanogaster and Streptomyces coelicolor (43 and 27% similarity, respectively), and partial EST sequences in pig, bull, rat, and Xenopus (not shown). All sequences are related by the presence of a conserved ATP-binding site, with the recognized subdomains (I–V) (13Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (701) Google Scholar). Remarkably, all sequences are similar in size, around 330 amino acids, except that of C. elegans, which contains an additional amino-terminal domain (1–192 amino acids) not shared by the other putative orthologues. Comparison with prokaryotic sequences by HMM search (10Durbin R. Eddy S. Krogh A. Mitchison G. The Theory Behind Profile HMMs: Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids. Cambridge University Press, Cambridge, UK1998Google Scholar) showed significant relationship with members of the ROK family containing sugar kinases (13Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (701) Google Scholar). Fig. 1 shows the amino acid sequence comparison between eukaryotic enzymes and the most representative members of the prokaryotic family, including the YHCI, YAJF, YCFX, and ALSK enzymes from E. coli and SCRK from Bacillus subtilis. These data strongly suggested that SIP16 is a eukaryotic sugar kinase. When preparing the first version of this manuscript, a final search in the data banks revealed that new sequences were included (NP_062415; NP_060037; CAB61849) with high similarity (98%) to mSIP16. These sequences corresponded to the murine and human GlcNAcK proteins (6Hinderlich S. Ber
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