tisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ..... Lrz PY J h ITP-ICJ. FIG. 3. aPY and aPTP-1C Western blots of fractions from.
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 267, No. 33, Issue of November 25, pp. 23447-23450,1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Ine. Printed in U.S. A.
Communication
receptor (I), the c-fmsproto-oncogeneproduct (2), which possesses tyrosine kinase activity (3). Incubation of rnacrophages with CSF-1 causes non-covalent CSF-1 receptor (CSF1R) dimerization, activation, andtyrosinephosphorylation followed by the tyrosine phosphorylationof several primarily cytoplasmic proteins (4-7). Theidentity of these proteins has not been established. Their appearance is maximally stimulated by 60 s at 37 "C,but differences in the kinetics of their (Received for publication, August 27, 1992) appearance can be resolved at 4 "C (6). They may be phosYee-GuideYeung, Karen L. Berg$, phorylated directly bythe activated CSF-IR or indirectly by Fiona J. Pixleys, Ruth Hogue Angeletti, and non-receptor tyrosine kinases that are activated as part of a E. Richard StanleyT signal transduction process, or their tyrosine phosphorylation From the Department ofDeuelopmental Biology and may increase due to growth factor-induced inhibition of a Cancer, Albert Einstein College of Medicine, protein tyrosine phosphatase. Bronx, New York 10461 As few of the tyrosine-phosphorylated proteins appear to stably associate with the CSF-1R: receptor-based purificaAn -64-kDa cytoplasmic protein is rapidly phosphorylated in tyrosine in the response of macrophages tion/cloning methods are not generally applicable. Therefore to colony stimulatingfactor-1. To identify this protein, we have adopted the approach of identifyingthem directly by -64-kDa a protein BAC1.2F6 macrophages were incubated with or with- purification and microsequencing. Because out colony stimulating factor-1, the phosphotyrosine- is markedly tyrosine-phosphorylated in macrophages in recontainingportion oftheir cytosolic fractionssubjected sponse to CSF-1, we havefocused our initialpurification efforts on the CSF-1-induced tyrosine-phosphorylated proto size exclusion chromatography, and the46-70-kDa fraction further fractionated by reverse phase high teins of 45-70 kDa. In this communication,we briefly describe pressure liquid chromatography (RP-HPLC). Tryptic methods for the purification ofthese proteins in sequenceable peptides of pooled RP-HPLCfractions from stimulated amountsandshow that the -64-kDa cytoplasmicprotein cells (containing the-64-kDa protein and an-64-kDa which is rapidly tyrosine-phosphorylated in responseto CSFprotein) and from unstimulated cells (containing the 1 is protein tyrosine phosphatase1C (PTP-IC). -64-kDa protein alone), were sequenced directly. All seven readable sequences of 8 sequenceable peptides EXPERIMENTALPROCEDURES present uniquely in the stimulatedfraction were presCell Culture, Protein Purification, and Sequencing-Cells of the ent in the sequence of the 81% homology 2 domain- BAC1.2F5 macrophage line were cultured in 100-mm tissue culture containing protein tyrosine phosphatase-lC (PTP-1C). dishes and stimulated with CSF-1 (human recombinant macrophage The identityof the -64-kDa protein was confirmed by colony stimulating factor, a gift from Chiron Corp.) at 4 "C in the Western blottingwith an antibody raised to aPTP-1C presence of 2 mM iodoacetic acid to increase the yield of phosphotypeptide.Therapid, growth factor-induced tyrosine rosine-containing proteins as described previously (4, 8). The cells were then washed once with ice-cold phosphate-buffered saline (136 phosphorylation of PTP-1C suggests that it may be involved in very early events in growth factor signal mM NaCI, 3 mM KCI, 8 mM NaZHPO,, 1.5 mM KHzP04,pH 7.4) (PBS), scraped in cold PBS containing 100 pM sodium orthovanadate transduction.
Protein Tyrosine Phosphatase-1C Is Rapidly Phosphorylatedin Tyrosine in Macrophages in Response to Colony Stimulating Factor- 1"
The action of the mononuclear phagocyte growth factor, colony stimulating factor-1 (CSF-1)l is mediated by aspecific * This work was supported by National Institutes of Health Grant CA 26504, Albert Einstein Core Cancer Grant P3O-CA 1330, and a grant from the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. $Supported by National Institutes of Health TrainingGrant 2 T32 CA09173. Fellow of the Leukemia Society of America. II To whom correspondence should be addressed Dept. of Developmental Biology and Cancer, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461. The abbreviations used are: CSF-1, colony stimulating factor-1; PY, phosphotyrosine; aPY, anti-phosphotyrosine; CSF-lR, colony stimulating factor-1 receptor; FPLC, fast protein liquid chromatography; PBS, phosphate-buffered saline; PTPase, protein tyrosine phosphatase; PTP-lC, protein tyrosine phosphatase 1C; RP-HPLC, reverse phase high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; SH2, src homology 2; TBS, Tris-buffered saline.
(Fisher Scientific), 100 p~ phenylmethylsulfonyl fluoride (Sigma) and 2 mM iodoacetic acid (Fluka), collected in centrifuge bottles, and pelleted at 400 X g for 4 min at 4 "C. Homogenization and the subcellular fractionation were performed as described by Yeung and Stanley (9). Phosphotyrosine (PY)-containing proteins from the cytosol of two thousand subconfluent cultures were prepared by affinity column chromatography using anti-phosphotyrosine (aPY) antibody (10) coupled to Sepharose 4B (Oncogene Science). The affinity chromatography was carried out with the buffer system ofLi et al. (8) except that 0.8% octyl glucoside (Boehringer Mannheim) was used instead of 0.5% Nonidet P-40 inthe last 3 washes of the column prior to elution and in the elution buffer. Proteins eluted with 5 mM phenyl phosphate (Sigma) were concentrated to approximately 500 pl by Centriprep 30 (Amicon) and then to100 pl by Centricon 30 (Amicon). Crystalline guanidine-HCI (110 mg) (Pierce Chemical Co.), 94 p1 of 2 M Tris-HC1 (Sigma), pH8.5, and 1.3 pl of P-mercaptoethanol (Pierce) were added to yield 190pl of a 6M guanidine-HC1,lOO mM Tris-HCI, 100 mM mercaptoethanol solution, pH 8.5. The proteins were reduced and denatured by incubation for 2 h at 20 "C and then overnight a t 4 "C. The denatured phosphotyrosyl proteins were separated by size exclusion chromatography on a Superose S-6 column (10 X 300 mm, Pharmacia) in an FPLC system (Pharmacia) in 6 M guanidine-HC1, 50 mM Tris-HC1,0.5% dodecyltrimethylammonium bromide (Sigma), and 100 mM mercaptoethanol, pH 6.5, at room temperature with a flow rate of 0.25 ml/min. Fractions (0.3 ml) were collected and the protein in each fraction detected by silver staining (11) of gradient
23447
K. L. Berg, unpublished results.
23448
CSF-1 Induces PTP-IC Tyrosine Phosphorylation in Macrophages
(7.5-17.5% acrylamide) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (12). Fractions containing proteins of interest were pooled and concentrated to 100 pl (Centricon 10, Amicon) a t 18 "C. The concentrate was diluted with 1 ml of 6 M guanidine-HCI, containing 0.8% octyl glucoside and concentrated again to 100 pl twice more a t 18 "C. It was then acidified with 20% trifluoroacetic acid (Pierce) to
