This action might not be possible to undo. Are you sure you want to continue?
1007/s10930-006-9015-6 Published Online: September 1, 2006
Expression and Puriﬁcation of Human PAG, a Transmembrane Adapter Protein Using an Insect Cell Expression System and its Structure Basis
In this study, we report the puriﬁcation and structure basis of human phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a C-SRC tyrosine kinase (CSK)-binding protein. Human PAG was produced using an insect cell expression system. The PAG was puriﬁed by metal aﬃnity, ion exchange, and gel ﬁltration chromatographies. The ﬁnal purity of gel-puriﬁed PAG was evaluated by SDS-PAGE and mass spectrometry. Recombinant human PAG migrates to 60 kDa on SDS-PAGE gel, while native PAG is a 46 kDa transmembrane adapter protein in lipid rafts. Recombinant human PAG has a diﬀerence of 2590.7 Da with a calculated mass (47803.41 Da) and an observed mass (50394.1 Da) by mass spectrometry. Consequently, although human PAG sequence shares well-known sites for modiﬁcations such as myristoylation, palmitoylation, and tyrosine phosphorylation sites, perhaps the diﬀerence suggests the existence of unknown modiﬁcation sites. We show the high PAG-binding ability with CSK in vitro as well as the human PAG structure characterized by 11 a-helix structures including a 3 kDa transmembrane domain.
KEY WORDS: SFKs; myocardial infarction; protein modiﬁcation; mass spectrometry; a-helix structure.
1. INTRODUCTION SRC family tyrosine kinases (SFKs) play important roles in ligand-induced cellular responses including proliferation, survival, adhesion, and migration. The kinase activation of SRC in myocardial infarction is associated with the expansion of infarct volume and the decline of cardiac function including ﬁbrosis and survival. Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) was found in lipid rafts as a SRC negative regulator or a C-SRC tyrosine kinase (CSK) activator (Brdicka et al., 2000; Kawabuchi et al., 2000; Takeuchi et al., 2000). This regulator may be appli-
cable as a novel approach for myocardial infarction treatment. In the present study, we describe its puriﬁcation method when recombinant human PAG was puriﬁed via metal aﬃnity, ion exchange and gel ﬁltration chromatographies. The ﬁnal purity of PAG was over 95% on SDS-PAGE and further evaluated by mass spectrometry. In addition to the puriﬁcation method, the present study shows a whole structure of the PAG with a modiﬁed 50394.1 Da mass variously, which human PAG is composed of eleven a-helix structures including a 3 kDa transmembrane domain. And also, we show the high binding ability between PAG and CSK shown in the presence of 1.5 M NaCl.
Department of Protein Research, ProstaColon, 85 NE, Takamatsu, Kahoku, Ishikawa, 929–1215, Japan. To whom correspondence should be addressed. E-mail: email@example.com
Abbreviations: SFKs, SRC family tyrosine kinases; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomains; CSK, C-SRC tyrosine kinase; GRP78, 78 kDa glucoseregulated protein; ORP150, 150 kDa oxygen-regulated protein.
1572-3887/06/0600-0295/0 Ó 2006 Springer Science+ Business Media, Inc.
1B) because human PAG sequence indeed has the highly conserved myristoylation.5% NP40. Mass Spectrometry and Modiﬁcation of PAG Its actual observed mass was 50394. We think that recombinant His-tagged PAG may migrate from 46 to 60 kDa on our analyzed SDS-PAGE gel (Fig.4.org/). The gel-puriﬁed PAG has a high purity of more than 95% as shown in Fig. and 1 mM 2-ME using the TALON metal resin (Clontech) column.1. In fact. MALDI-TOF (MS) of PAG Mass spectra of gel-puriﬁed PAG were obtained using a Voyager-DE STR BiospectrometryTM Workstation (PerSeptive Biosystems Inc. Sephacryl S-400 HR) chromatographies. 1A). 1994. and the human were aligned using the DDBJ CLUSTALW 1.2. 3B.. Abundant acidic residues within protein sequence aﬀect migration of the protein on SDSPAGE gel. 2. 0. protein modiﬁcation (e. Miyazaki et al. the rat.41 Da (Fig. 1982) on ExPASy server (http:// www.3. no matching with the calculated mass. After metal aﬃnity and ion exchange chromatographies. 0. SDS-Page Analysis of PAG Human PAG was overexpressed in Sf9 insect cells after recombinant baculovirus infection (Fig. active fractions were further gel-chromatographed using the same column under the diﬀerent NaCl concentrations ranging from 0.1. 1994). The sequences of PAGs from the mouse. Native PAG is an approximately 46 kDa transmembrane adapter protein located in lipid rafts. 1). 2. Recombinant Baculovirus Construction. CSK-binding Assay We previously described a puriﬁcation method of recombinant human CSK (in submission).br. The PAG was eluted when imidazole was reached to a 0.5 M NaCl. 1 ml of PAG solution was mixed with a fresh sinapinic acid matrix in 40% acetonitrile including 0. Hydropathy Proﬁling.15 to 1. and phosphorylation etc. Recombinant Bacmid-PAG was transfected into Sf9 insect cells using the CELLFECTIN reagent based on the instruction manual for the Bac-to-Bac baculovirus expression system (Invitrogen).g.5 M (buﬀers: 50 mM Tris pH7. A diﬀerence with the .5% NP40. 1B. Moreover. 3.4. 0.296 2. 0. coli cells.15$1. Secondary Structure Prediction. which however migrates anomalously as an $80 kDa protein on SDSPAGE gels. and tyrosine phosphorylation sites shown in Fig. and 1 mM 2-ME) via ion exchange (Bio-Rad. imidazole ($0. palmitoylation.4 M). Sf9 insect cells were infected with recombinant baculovirus during a logphase of the cells..5 M NaCl.2. we mixed Sf9 insect cells expressed the CSK with Sf9 insect cells expressed PAG. RESULTS AND DISCUSSION 3. and then recombinant human PAG was expressed. Sf9 insect cell lines were maintained in spinner ﬂasks (100 ml culture) at 27°C from 30 Â 104/ml to 250 Â 104/ml for 3–4 days in IPL41 medium (Invitrogen) including 10% FBS. 2). recombinant His-tagged PAG was approx. and Sequence Alignment of PAG(s) A putative secondary structure of human PAG was obtained using the SOPM secondary structure prediction tool (Geourjon and Deleage.1 Da. Recombinant human PAG was produced according to the above manual as follows. High Q Econo-Pac) and gel ﬁltration (Amersham. 2004). while a calculated mass of recombinant His-tagged PAG was 47803.4. A hydropahy proﬁle of human PAG sequence was Takeuchi obtained using the Kyte-Doolittle tool (Kyte and Doolittle. and 1 mM 2-ME).5% NP40. glucosylation. palmitoylation. N-His) was cloned into donor vector pFastBac HTa and recombinant pFasBac HTaPAG was transformed into competent DH10Bac E. EX-T0343-I01.4) including 0.1% triﬂuoroacetic acid on a MALDI plate. Recombinant His-tagged PAG was puriﬁed in 50 mM Tris buﬀer (pH7.2 M concentration.. 60 kDa on our analyzed SDS-PAGE gel (Fig. myristoylation. In this assay. MA. USA).83 program (Thompson et al. The collected active fractions were further puriﬁed in a ﬁnal buﬀer (50 mM Tris pH7.15 M NaCl. MATERIALS AND METHODS 2. 0. 10% glycerol..expasy. 2. Expression and Puriﬁcation of PAG The cDNA encoding human PAG (RZPD. 3.) is also similar.
The masses observed are shown. each including a short extracellular domain ($13aa).. and phosphorylation (80 Da Â 5) to a calculated mass of recombinant His-tagged PAG. (A) Putative secondary structure (top) and hydropathy proﬁle (bottom) of human PAG were obtained using the SOPM secondary structure prediction and Kyte-Doolittle tools. Even if we add the known modiﬁed masses of myristoylation (210 Da Â 1). which are potential sites for phosphorylation (Fig. calculated and observed masses of the PAG is 2590. a transmembrane domain (27aa). (B) Sequence alignment of PAGs from the mouse. Representative known modiﬁcation sites are as follows. From a result of our mass spectrometry.7 Da. respectively.Expression and Puriﬁcation of Human PAG 297 Fig. SDS-PAGE analysis of the expressed and gel-puriﬁed PAGs. Mass spectrometric analysis of gel-puriﬁed PAG. The human PAG shows over 90% similarity with the mouse and the rat in the amino acid sequence. Purity of gel-puriﬁed PAG was ﬁnally evaluated by mass spectrometry. These PAGs are also characterized by a common C-terminal TRL sequence which interacts with an N-terminal PDZ domain of ERM-binding phosphoprotein EBP50 (Fig. Identical residues were hatched. Human PAG shares ten tyrosine residues within its cytoplasmic domain sequence.7 Da) is unknown. lane 2: PAG expression in Sf9 insect cells (crude extract). This process involves a change in the phosphorylation status of downstream proteins by modiﬁcation of speciﬁc tyrosine or serine/threonine residues. palmitoylation (238 Da Â 2). a mouse (GenBank Accession No. Molecular structure of PAG. 3B). bolds. and the human were performed using the program DDBJ CLUSTALW 1. (B) Analysis of gel puriﬁcation step of PAG. and a human (AF240634). and a large cytoplasmic domain. catalyzed by diﬀerent kinases. the rat. we think that perhaps the PAG shares the unknown modiﬁcation sites including potential modiﬁcation sites for insect endogeneous kinases because an observed mass of the human PAG expressed in Sf9 insect cells is larger than its predicted mass. The phosphorylation of proteins. 2001. 3.3. 2002). lane 1: standard molecular weight marker. nine of which are potential phosphorylation sites by SFKs (YXXV/I/L). Q3U1F9). PAG is a 429 amino acid (aa) in the mouse. Itoh et al. 2. the remaining diﬀerence (1504. PAG also contains many threonine and serine residues. Putative transmembrane domains were boxed. .. is critical to cellular functions associated with signal transduction. The N-terminal myristoylation (glycine). PAG Structure The 3D structure of PAG is not yet clariﬁed. Fig. 3. and phosphorylation (tyrosine) sites were shown with asterisks. 1. The following sequences were used for the alignment. a rat (Q9JM80). (A) Lane 1: standard molecular weight marker. 3B) (Brdickova et al. and 432aa in the human. other lanes: gel-puriﬁed PAGs.83. palmitoylation (cysteine). 424aa in the rat. and arrow heads. These ﬁndings suggest that mammalian PAGs have a highly conserved c Fig.
298 Takeuchi .
A.. Redﬁeld. Kinoshita. Nada. Yanagi. in which GRP78 (78 kDa glucose-regulated protein). (2001). Y. B.. PAG-CSK complex formation. J. Interestingly. Biol. K.5 M NaCl (Fig. H. S... M. K. Nakajima. Imuta. Bruyns. S.. Surprisingly. (1994).. Y. 3. Vidal. Immunol.. N. Leo.. K. cancer. N.. Biol. previous REFERENCES Brdicka. Kyte.. Currently. Nucleic Acids Res. Chem. Miyazaki. Shevchenko. we have functionally and structurally characterized human PAG as a SFK negative regulator. Scherer. 201: 1548–1553. M. Res.. PAG-CSK Complex Formation Under High Ionic Strength Condition Previous studies have shown a strong interaction between PAG and CSK (Kawabuchi et al. M. Hilgert. T. J. Korinek. Yamasaki. Toledo. we are studying relations between ORP150 and activity regulation of SFKs with PAG. Thompson. Andera. and Brot. Milgram. 3D structure.4. it is insuﬃcient to understand the functions of SFKs in cardiac infarction and insuﬃciency as well as the identiﬁcation of potential modiﬁcation sites of PAG and PAG-catalytic kinases from insect. Spicka. Biochem. Nucleic Acids Res. A. . M. J. D31–D34. ORP150 is speciﬁcally induced by hypoxia stress (Kuwabara et al. sheet: 60. other lanes: gelpuriﬁed PAG-CSK complexes. and Tateno. S. (1996). Y. (1994).. 275: 29183–29186. T. J. A.. D.. G.. H. Ikeo. We show the human PAG structure which is composed of eleven a-helix structures including a 3 kDa transmembrane domain clariﬁed by hydropahy proﬁling (Fig. Nature 404: 999–1003. Geourjon.. Sakakibara. K. B. 157: 105–132. Ikeda.. T. Carlino.. In the present study. DQ350134 and DQ372932) including transcript variant from cardiac tissues with infarction. Inappropriate or deregulated phosphorylation is deeply associated with various diseases such as diabetes. A.. Kornacker.. neurological and autoimmune diseases.. V. Racker. Kuwabara. N.... However.. and Kamada. A.. regulates c-SRC kinase activity in vitro (Carlino et al.. and Saito. P. Kawabuchi. B. and Horejsi... Abdel-Ghany. 4. C. Tamura. Brdicka.... an ER chaperone. Itoh. Kuramitsu. Stern. M. We investigated a complex formation of PAG-CSK under the various concentrations of NaCl ranging from 0. Takayama. J. T. D. K.5 M NaCl. this study showed that PAG and CSK were able to maintain their complex formation under a high ionic strength condition of 1. O. A clear understanding of phosphorylation associated with a speciﬁc signaling pathway is the key to understanding pathway activation. V. (1994). Takeuchi.. (2004).. Gojobori. FEBS Lett. D... Takao. T. Y. Chem. and Doolittle.. we could not ﬁnd a similar structure to human PAG from the protein structure similarity search. Nagai. 271: 5025–5032. Cerny. (2000). N. (2000). V.9%. S.. P.. T. PAG and CSK were able to maintain their complex formation in the presence of 1. M. related diseases and an ultimate approach. J..3%. J..Expression and Puriﬁcation of Human PAG 299 study showed a relation between SFK and ER chaperone. Biol.. (1982). 2000)... Biophys. D. Miyazaki.. 191: 1591–1604. Angelisova. Strassman. Shimonishi. J. A. E. K. Mol. Commun. Brdickova. Ogawa. 22: 4673–4680. J. and Okada.. T.. J. 1996) and shows chaperone function in the ER. 507: 133– 136.. M. Tarakhovsky.. Horejsi. 4). Ogawa. Takeuchi... Fig. Protein Eng. (2000). J. Unfortunately. Presently. and Deleage. 3A). H. T. I.... lane 1: standard molecular weight marker. L. and Gibson.15 to 1. E. (2002). 32:(Database issue).. S.. Higgins. J. Y. and heart disease including cardiac insuﬃciency. Y. Satomi.. F...... H.. Kitagawa.. H. Angelisova. We have recently cloned two 150 kDa oxygen-regulated protein (ORP150) cDNAs (GenBank Accession No. and Schraven.5 M NaCl. Pavlistova. 1994). R. Weissbach. M. and Okada.. Arase. Hori. V. 168: 541–544. G. Sugawara. Maeda. L. 7: 157–164. S. Okada.. M. Matsumoto. Drbal. K.. 1994). and coil: 74%) (Geourjon and Deleage. Med. J.. Exp. we predicted a secondary structure of human PAG using the above tool with an entire accuracy of 70% (helix: 66..