Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKβ, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for β1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells. Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKβ, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for β1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells.
Afin d’evaluer un systeme de perfusion assiste par ordinateur grâce a un essai clinique, une methode de dosage plasmatique du propofol a ete validee, comprenant une extraction liquide/liquide, une separation par CLHP et une detection fluorimetrique. Le thymol a ete employe comme etalon interne. Le coefficient d’extraction moyen etait de 93 %. Cette technique est lineaire (Y = 0,289X + 0,0084, r = 0,9989), exacte, sensible (limite de detection = 10 ng/ml) et selective (temps de retention moyens du thymol et du propofol : 6,48 min et 10,73 min), la resolution est correcte, mais l’extraction est longue et delicate. Les medicaments employes au cours de l’essai clinique prevu (flunitrazepam, fentanyl et vecuronium) ont ete testes, sans causer d’interference analytique. La validation de cette methode a permis l’inclusion de 28 patients, devant subir une pose de prothese totale de hanche, qui ont recu du propofol administre par ce systeme. L’analyse des resultats est en cours.
Abstract Despite the efficacy of natalizumab in Multiple Sclerosis (MS) treatment, approximately 30% of patients do not respond favorably. Individual heterogeneity of T-cell response to VLA-4 natalizumab-mediated blockade may underlie disparities in treatment efficacy. Here, a high-content cell imaging (HCI) pipeline was implemented to profile the in vitro effects of natalizumab on VLA-4-stimulated leukocytes from MS patients prior to treatment. Unsupervised clustering of image data partially discriminated non-responder MS patients based on morphology, F-actin organization, and signaling-related features in CD8+ T cells. Treatment response was assessed through a Random Forest approach with predictive performance of 91% for a discovery cohort and 70% for a validation cohort. Unfavorable treatment response was associated with the inefficacy of natalizumab to impair the ability of pretreated CD8+ T cells to spread over VCAM-1. Our study unveils that CD8+ T cells from individual MS patients display heterogeneous susceptibility to natalizumab in vitro and highlights the potential of HCI-based pretreatment monitoring to assist individualized treatment prescription.
INSERM U275, LOA-ENSTA-Ecole Polytechnique, Palaiseau, DéApartement d'Anesthesie, HÔcpital de la Pitié, Paris, Laboratoire de Toxicologie, Hôpital Henri-Mondor, Créteil, Brigade des Sapeurs Pompiers de Paris, Paris, Clinique Toxicologlque, Hôpital Fernand Widal, Paris, and the G. E. F. I. (Groupe d'Etude des Fumées d'Inoendie), Paris, FRANCE.
The Crk-associated substrate p130Cas (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as "docking" molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either β1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56Lyn immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an in vitro binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of β1 integrin or BCR on human B cells. The Crk-associated substrate p130Cas (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as "docking" molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either β1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56Lyn immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an in vitro binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of β1 integrin or BCR on human B cells. INTRODUCTIONThe integrin family of adhesion molecules are involved in transducing biochemical signals into the cell, resulting in diverse biological events. Among these signals are tyrosine phosphorylations of specific proteins such as the focal adhesion kinase p125FAK (Fak) 1The abbreviations used are: FAKp125FAKCasp130CasHEF1human enhancer of filamentation 1SHsrc homologyBCRB cell antigen receptorRAFTKrelated adhesion focal tyrosine kinaseCblp120c-CBLRAMrabbit anti-mouse IgCBcytochalasin Banti-P-tyranti-phosphotyrosineIVKin vitro kinase assayPAGEpolyacrylamide gel electrophoresis. (1Schwartz M. Shaller M. Ginsberg M. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar). Integrin cytoplasmic domains are associated with actin-containing cytoskeleton components, and one concept of integrin-mediated tyrosine phosphorylations is that oligomerization of integrins reorganizes the cytoskeleton into a framework that supports interactions between components of the intracellular signaling machinery (2Shattil S. Ginsberg M. Brugge J. Curr. Opin. Cell Biol. 1994; 6: 695-704Crossref PubMed Scopus (188) Google Scholar). In support of this hypothesis is the observation that inhibitors of cytoskeletal assembly also inhibit integrin-mediated tyrosine phosphorylations (3Huang M. Lipfert L. Cunningham M. Brugge J. Ginsberg M. Shattil S. J. Cell Biol. 1993; 122: 473-483Crossref PubMed Scopus (155) Google Scholar).B lymphocytes express several different integrins that are involved in cell localization within specific microenvironments (4Freedman A.S. Munro M.J. Rice G.E. Bevilacqua M.P. Morimoto C. McIntyre B.W. Rhynhart K. Pober J.S. Nadler L.M. Science. 1990; 249: 1030-1033Crossref PubMed Scopus (229) Google Scholar, 5Koopman G. Parmentier H.K. Schuurman H.-K. Newman W. Meijer C.J.L.M. Pals S. J. Exp. Med. 1991; 173: 1297-1304Crossref PubMed Scopus (249) Google Scholar). Ligation of integrins on pre-B and mature B cells appears to be involved in regulating cell survival (6Ryan D.H. Nuccle B.L. Abboud C.N. Liesveld J.L. J. Immunol. 1990; 145: 477-484PubMed Google Scholar, 7Ryan D. Nuccie B. Abboud C. Winslow J. J. Clin. Invest. 1991; 88: 995-1004Crossref PubMed Scopus (186) Google Scholar, 8Ryan D.H. Nuccie B.L. Abboud C.N. J. Immunol. 1992; 149: 3759-3764PubMed Google Scholar, 9Koopman G. Keehnen R. Lindhout E. Newman W. Shimuzu Y. Van Seventer G. De Groot C. Pals S. J. Immunol. 1994; 152: 3760-3767PubMed Google Scholar). The identification of proteins that are tyrosine phosphorylated following integrin ligation is important to understanding how integrin-mediated signaling regulates B cell function. We have previously reported the prominent tyrosine phosphorylation of proteins of 105 to 130 kDa following β1 integrin cross-linking on human B cells (10Freedman A. Rhynhart K. Nojima Y. Svahn J. Eliseo L. Benjamin C. Morimoto C. Vivier E. J. Immunol. 1993; 150: 1645-1652PubMed Google Scholar, 11Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar). Two of these substrates have been identified as Fak (11Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar) and p120c-CBL (Cbl) (12Manié S. Sattler M. Astier A. Phifer J.S. Canty T. Morimoto C. Druker B. Salgia R. Griffin J.D. Freedman A.S. Exp. Hematol. 1996; Google Scholar), the cellular homologue of the oncogene v-CBL.Following integrin ligation in fibroblasts, another tyrosine phosphorylated protein known as p130Cas (Cas) has been identified (13Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 14Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 15Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). Integrin-mediated homotypic adhesion in a B cell line also induced tyrosine phosphorylation of Cas (16Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Cas (rk ssociated ubstrate) was originally described as a major tyrosine phosphorylated protein in v-crk- or v-src-transformed cells (17Kanner S. Reynolds A. Wang H.-C.R. Vines R. Parsons J. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yakazi Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Cas is an SH3 domain containing molecule with 15 potential Crk-SH2-binding motifs and several potential binding motifs for SH3 domains, suggesting that it may act as a "docking molecule" in intracellular signal transduction. In fact, Cas forms stable complexes with the SH2 domains of v-crk family members and v-src (16Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 17Kanner S. Reynolds A. Wang H.-C.R. Vines R. Parsons J. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yakazi Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 19Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar, 20Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 21Burnham M. Harte M. Richardson A. Parsons J. Bouton A. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar) and with Fak through binding to the SH3 domain of Cas (22Polte T. Hanks S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 23Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Recently, a Cas-related protein known as HEF1 (human enhancer of filamentation 1) 2During the time of revision of this manuscript, Minegishi, M., Tachibana, K., Sato, T., Iwata, S., Nojima, Y., and Morimoto, C. (1996) J. Exp. Med. 184, 1365, reported the cloning of Cas-L, which is identical to HEF1, and is tyrosine phosphorylated following β1 integrin ligation in T cells. has been isolated and characterized (24Law S. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). Analogous to Cas, HEF1 contains an SH3 domain and multiple Crk-SH2 binding motifs, associates with Fak and v-abl, and localizes to focal contacts. However, in contrast to Cas, HEF1 localizes to the cell nucleus, suggesting that Cas and HEF1 may have distinct functions in cell signaling.In the present report, we show a significant increase in the tyrosine phosphorylation of Cas and HEF1 induced by β1 integrin ligation on normal or transformed human B cells, with HEF1 being the predominant substrate. Ligation of the B cell antigen receptor (BCR) also induced tyrosine phosphorylation of predominantly HEF1, and similar to integrins, BCR-mediated HEF1 phosphorylation was dependent upon an intact actin network. We further showed that Cas and HEF1 complexed in vivo with the related adhesion focal tyrosine kinase RAFTK, the adapter protein CrkL, and Lyn kinase, indicating that both molecules may play an important role in B cell signaling.DISCUSSIONRegulation of B cell survival within specific microenvironments involves integrin engagement (6Ryan D.H. Nuccle B.L. Abboud C.N. Liesveld J.L. J. Immunol. 1990; 145: 477-484PubMed Google Scholar, 7Ryan D. Nuccie B. Abboud C. Winslow J. J. Clin. Invest. 1991; 88: 995-1004Crossref PubMed Scopus (186) Google Scholar, 8Ryan D.H. Nuccie B.L. Abboud C.N. J. Immunol. 1992; 149: 3759-3764PubMed Google Scholar, 9Koopman G. Keehnen R. Lindhout E. Newman W. Shimuzu Y. Van Seventer G. De Groot C. Pals S. J. Immunol. 1994; 152: 3760-3767PubMed Google Scholar). We previously reported that integrin-mediated signaling pathways in B cells regulates a cascade of tyrosine phosphorylation events (10Freedman A. Rhynhart K. Nojima Y. Svahn J. Eliseo L. Benjamin C. Morimoto C. Vivier E. J. Immunol. 1993; 150: 1645-1652PubMed Google Scholar, 11Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar). In the present study, we have determined that p130Cas and the Cas-like molecule p105HEF1 are expressed in B cells and that β1 integrin ligation or BCR engagement on human B cells promoted tyrosine phosphorylation principally of HEF1. Furthermore, HEF1 and Cas phosphorylation following both stimuli appeared to be closely linked with cytoskeletal organization, and we identified several signaling molecules, including p53/56Lyn kinase, RAFTK, and CrkL, associated both with Cas and HEF1.HEF1 was cloned from a HeLa cDNA library, which when expressed in Saccharomyces cerevisiae, strongly enhanced pseudohyphal growth, suggesting a role for HEF1 in regulating cell signaling and morphology (24Law S. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). Although HEF1 RNA was present in all tissues examined, the highest levels were in placenta, lung, and kidney. HEF1 is 64% similar to Cas at the amino acid level. Both proteins have multiple potential SH2 binding sites and a striking similarity in the SH3 domain and the C terminus. This raises the question as to why B cells express two very similar proteins. In tonsillar B cells as well as in B cell lines, both HEF1 and Cas were present with the exception of the pre-B cell line Nalm-6, which did not express Cas. β1 integrin-mediated tyrosine phosphorylation was mainly detected in HEF1 but not in Cas, except in the more terminally differentiated B cell line ARH-77. Generally, the p105 rather than the p120 form of HEF1 was the predominant species seen and tyrosine phosphorylated. Similarly, HEF1 rather than Cas was phosphorylated following BCR ligation in tonsillar B cells, however Cas could be phosphorylated under BCR ligation in the surface IgG positive cell lines, ARH-77 and SB. 3S. N. Manie, A. Astier, and A. S. Freedman, unpublished data. Therefore, Cas appears to be phosphorylated only in more terminally differentiated cells, which suggests that Cas and HEF1 may have distinct functions depending on the differentiated state of the cell. Cellular localization studies provide further evidence for distinct functions of HEF1 and Cas and with Cas present at focal contacts, whereas HEF1 localizes to the cell periphery and the nucleus (24Law S. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar).Similar to Cbl (12Manié S. Sattler M. Astier A. Phifer J.S. Canty T. Morimoto C. Druker B. Salgia R. Griffin J.D. Freedman A.S. Exp. Hematol. 1996; Google Scholar, 26Cory G. Lovering R. Hinshelwood S. MacCarthy-Morrogh L. Levinsky R. Kinnon C. J. Exp. Med. 1995; 182: 611-615Crossref PubMed Scopus (126) Google Scholar), HEF1 is a common substrate in B cells for both integrin and antigen receptors. However, tyrosine phosphorylation of HEF1 and Cbl in fact differ in BCR-mediated signaling pathways. The kinetics of HEF1 phosphorylation was slower than that of Cbl. HEF1 phosphorylation was reduced by prior treatment of cells with cytochalasin B, whereas Cbl phosphorylation was not affected. These findings also suggest that BCR-mediated HEF1 phosphorylation correlated with actin filament reorganization. Interestingly, BCR ligation initiates microfilament assembly (31Melamed I. Downey G. Aktories K. Roifman C. J. Immunol. 1991; 147: 1139-1146PubMed Google Scholar) and induces a redistribution of signaling molecules such as ras (32Graziadei L. Riabowol K. Bar-Sagi D. Nature. 1990; 347: 396-400Crossref PubMed Scopus (72) Google Scholar) and neurofibromin (33Boyer M. Gutmann D. Collins F. Bar-Sagi D. Oncogene. 1994; 9: 349-357PubMed Google Scholar), which is inhibited by cytochalasin. Hence, analogous to integrin-mediated tyrosine phosphorylation (2Shattil S. Ginsberg M. Brugge J. Curr. Opin. Cell Biol. 1994; 6: 695-704Crossref PubMed Scopus (188) Google Scholar), some aspects of BCR-mediated signal transduction may require that a functional cytoskeleton serve as a framework that regulates the efficiency of interactions between signaling molecules and allows tyrosine phosphorylation of compartmentalized cellular proteins.The structure of Cas and HEF1 includes several SH2-binding motifs that are similar to the consensus binding motif for the Crk SH2 domain (34Songyang Z. Shoelson S. Chudhuri M. Gish G. Pawson T. Haser W. King F. Roberts T. Neel B. Birge R. Fajardo J. Chou M. Hanafusa H. Schaffhausen B. Cantley L. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). We showed here that both Cas and HEF1 bind to CrkL. Furthermore, all tyrosine phosphorylated Cas and HEF1 associate with CrkL, and this interaction is mediated by the SH2 domain of CrkL. 4A. Astier, S. N. Manie, S. F. Law, T. Canty, N. Haghayeghi, B. J. Druker, R. Salgia, E. A. Golemis, and A. S. Freedman, submitted for publication. Since the CrkL SH3 domain has been reported to bind to two guanine nucleotide exchange factors, C3G and mSOS, Cas and HEF1 might provide potential important links of β1 integrin and BCR signaling to the ras and or Rap1 pathways (35Matsuda M. Hashimoto K. Muroya K. Hasegawa H. Kurata T. Tanaka S. Nakamura S. Hattori S. Mol. Cell. Biol. 1994; 14: 5495-5500Crossref PubMed Scopus (183) Google Scholar, 36ten Hoeve J. Cancer Res. 1994; 54: 2563-2567PubMed Google Scholar). Therefore, by participating in a multimolecular complex formation, Cas and HEF1 may propagate downstream signals.The focal adhesion kinase Fak can associate with both Cas and HEF1 (22Polte T. Hanks S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 24Law S. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). These interactions are mediated by the highly homologous SH3 domains of Cas and HEF1, associating with the polyproline SH3 binding motif in Fak. We observed an in vivo association between the related adhesion focal tyrosine kinase RAFTK with Cas and HEF1. The interaction of Cas and HEF1 with RAFTK is also likely to be mediated through the SH3 domains of Cas and HEF1, binding to the C-terminal polyproline motif of RAFTK that is identical to that present in Fak. Since RAFTK is expressed in certain B cell lines independently of Fak and is phosphorylated under integrin and BCR stimulation (29Astier A. Avraham H. Manie S. Groopman J. Druker B. Canty T. Avraham S. Freedman A. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), the associations of Cas and HEF1 with RAFTK may be important in these signaling pathways.In contrast to HEF1, Cas contains a C-terminal proline-rich region that is an Src-SH3 binding motif (25Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). We have shown in an in vitro binding assay using a GST-fusion protein containing the C-terminal proline-rich region of Cas that p53/56Lyn, p59Fyn, and p59/62Hck, but not p55Blk or p56Lck, could bind to this motif. In addition, we demonstrated the presence of Cas in Lyn immunoprecipitates, whereas Cas was only weakly detected in Fyn immunoprecipitates and not in Hck immunoprecipitates. The anti-Fyn and anti-Lyn antibodies were raised against similar regions of the two molecules, allowing the comparison between them. The anti-Hck antibody was raised against a different region of the kinase, and we can not exclude the possibility that this antibody may have interfered with Cas binding. The interaction of Cas with Lyn in vivo likely occurred through the SH3 domain of Lyn because (i) a GST-fusion protein mutated in the C-terminal proline-rich region of Cas was unable to precipitate Lyn, and (ii) Cas phosphorylation following integrin or BCR ligation only minimally increased the presence of Cas in Lyn immunoprecipitates (not shown). HEF1 was also present in Lyn immunoprecipitates. However, HEF1 does not possess the src-SH3 binding motif that is present in Cas. Similar to Cas, HEF1 phosphorylation did not significantly increase its association with Lyn (not shown). Whether HEF1 can associate with Lyn through a non-canonical SH3 binding motif is under investigation. Alternatively, the C-terminal region of Cas may be capable of mediating heterodimerization with HEF1 (24Law S. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar), and therefore, the Lyn immunoprecipitation may involve a ternary Lyn·Cas·HEF1 complex. Lyn kinase activity is stimulated following both BCR ligation (30Yamanashi Y. Kakiuchi T. Mizuguchi J. Yamamoto T. Toyoshima K. Science. 1991; 251: 192-194Crossref PubMed Scopus (335) Google Scholar) and as shown here following β1 integrin ligation, suggesting that Lyn may be a kinase for Cas/HEF1. In support of this is that Lyn kinase could phosphorylate Cas/HEF1 in vitro when mixed prior to the kinase assay. Whether Cas/HEF1-associated Lyn is activated and responsible for Cas/HEF1 phosphorylation during the process of integrin or BCR ligation remains to be determined. Cas/HEF1-associated Lyn may also be involved in the phosphorylation of other molecules recruited by Cas/HEF1.The cytoskeletal dependence for HEF1 phosphorylation following integrin or BCR engagement on normal tonsillar B cells raises the possibility that HEF1 could integrate signals from both receptors. In T cells, signals from integrin and T cell antigen receptor have synergistic effects on proliferation (37Schimizu Y. Seventer G. Horgan K. Shaw S. J. Immunol. 1990; 145: 59-67PubMed Google Scholar, 38Matsuyama T. Yamada A. Kay J. Yamada K.M. Akiyama S.K. Schlossman S.F. Morimoto C. J. Exp. Med. 1989; 170: 1133-1148Crossref PubMed Scopus (246) Google Scholar, 39O'Rourke A. Mesher M. Nature. 1992; 358: 253-255Crossref PubMed Scopus (93) Google Scholar). Similarly, there is evidence for a functional cross-talk between integrins and BCR from studies of ligation of both receptors, where there appears to be modulation of normal B cell proliferation.3 Future studies will be directed toward understanding the function of HEF1 in integrin and BCR signaling pathways and gaining insight into the association of adhesion with antigen-induced activation of B cells. INTRODUCTIONThe integrin family of adhesion molecules are involved in transducing biochemical signals into the cell, resulting in diverse biological events. Among these signals are tyrosine phosphorylations of specific proteins such as the focal adhesion kinase p125FAK (Fak) 1The abbreviations used are: FAKp125FAKCasp130CasHEF1human enhancer of filamentation 1SHsrc homologyBCRB cell antigen receptorRAFTKrelated adhesion focal tyrosine kinaseCblp120c-CBLRAMrabbit anti-mouse IgCBcytochalasin Banti-P-tyranti-phosphotyrosineIVKin vitro kinase assayPAGEpolyacrylamide gel electrophoresis. (1Schwartz M. Shaller M. Ginsberg M. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar). Integrin cytoplasmic domains are associated with actin-containing cytoskeleton components, and one concept of integrin-mediated tyrosine phosphorylations is that oligomerization of integrins reorganizes the cytoskeleton into a framework that supports interactions between components of the intracellular signaling machinery (2Shattil S. Ginsberg M. Brugge J. Curr. Opin. Cell Biol. 1994; 6: 695-704Crossref PubMed Scopus (188) Google Scholar). In support of this hypothesis is the observation that inhibitors of cytoskeletal assembly also inhibit integrin-mediated tyrosine phosphorylations (3Huang M. Lipfert L. Cunningham M. Brugge J. Ginsberg M. Shattil S. J. Cell Biol. 1993; 122: 473-483Crossref PubMed Scopus (155) Google Scholar).B lymphocytes express several different integrins that are involved in cell localization within specific microenvironments (4Freedman A.S. Munro M.J. Rice G.E. Bevilacqua M.P. Morimoto C. McIntyre B.W. Rhynhart K. Pober J.S. Nadler L.M. Science. 1990; 249: 1030-1033Crossref PubMed Scopus (229) Google Scholar, 5Koopman G. Parmentier H.K. Schuurman H.-K. Newman W. Meijer C.J.L.M. Pals S. J. Exp. Med. 1991; 173: 1297-1304Crossref PubMed Scopus (249) Google Scholar). Ligation of integrins on pre-B and mature B cells appears to be involved in regulating cell survival (6Ryan D.H. Nuccle B.L. Abboud C.N. Liesveld J.L. J. Immunol. 1990; 145: 477-484PubMed Google Scholar, 7Ryan D. Nuccie B. Abboud C. Winslow J. J. Clin. Invest. 1991; 88: 995-1004Crossref PubMed Scopus (186) Google Scholar, 8Ryan D.H. Nuccie B.L. Abboud C.N. J. Immunol. 1992; 149: 3759-3764PubMed Google Scholar, 9Koopman G. Keehnen R. Lindhout E. Newman W. Shimuzu Y. Van Seventer G. De Groot C. Pals S. J. Immunol. 1994; 152: 3760-3767PubMed Google Scholar). The identification of proteins that are tyrosine phosphorylated following integrin ligation is important to understanding how integrin-mediated signaling regulates B cell function. We have previously reported the prominent tyrosine phosphorylation of proteins of 105 to 130 kDa following β1 integrin cross-linking on human B cells (10Freedman A. Rhynhart K. Nojima Y. Svahn J. Eliseo L. Benjamin C. Morimoto C. Vivier E. J. Immunol. 1993; 150: 1645-1652PubMed Google Scholar, 11Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar). Two of these substrates have been identified as Fak (11Manié S. Astier A. Wang D. Phifer J. Chen J. Lazarovits A. Morimoto C. Freedman A. Blood. 1996; 87: 1855-1861Crossref PubMed Google Scholar) and p120c-CBL (Cbl) (12Manié S. Sattler M. Astier A. Phifer J.S. Canty T. Morimoto C. Druker B. Salgia R. Griffin J.D. Freedman A.S. Exp. Hematol. 1996; Google Scholar), the cellular homologue of the oncogene v-CBL.Following integrin ligation in fibroblasts, another tyrosine phosphorylated protein known as p130Cas (Cas) has been identified (13Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 14Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 15Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). Integrin-mediated homotypic adhesion in a B cell line also induced tyrosine phosphorylation of Cas (16Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Cas (rk ssociated ubstrate) was originally described as a major tyrosine phosphorylated protein in v-crk- or v-src-transformed cells (17Kanner S. Reynolds A. Wang H.-C.R. Vines R. Parsons J. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yakazi Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar). Cas is an SH3 domain containing molecule with 15 potential Crk-SH2-binding motifs and several potential binding motifs for SH3 domains, suggesting that it may act as a "docking molecule" in intracellular signal transduction. In fact, Cas forms stable complexes with the SH2 domains of v-crk family members and v-src (16Petruzzelli L. Takami M. Herrera R. J. Biol. Chem. 1996; 271: 7796-7801Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 17Kanner S. Reynolds A. Wang H.-C.R. Vines R. Parsons J. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar, 18Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yakazi Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (592) Google Scholar, 19Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar, 20Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 21Burnham M. Harte M. Richardson A. Parsons J. Bouton A. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar) and with Fak through binding to the SH3 domain of Cas (22Polte T. Hanks S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 23Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Recently, a Cas-related protein known as HEF1 (human enhancer of filamentation 1) 2During the time of revision of this manuscript, Minegishi, M., Tachibana, K., Sato, T., Iwata, S., Nojima, Y., and Morimoto, C. (1996) J. Exp. Med. 184, 1365, reported the cloning of Cas-L, which is identical to HEF1, and is tyrosine phosphorylated following β1 integrin ligation in T cells. has been isolated and characterized (24Law S. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). Analogous to Cas, HEF1 contains an SH3 domain and multiple Crk-SH2 binding motifs, associates with Fak and v-abl, and localizes to focal contacts. However, in contrast to Cas, HEF1 localizes to the cell nucleus, suggesting that Cas and HEF1 may have distinct functions in cell signaling.In the present report, we show a significant increase in the tyrosine phosphorylation of Cas and HEF1 induced by β1 integrin ligation on normal or transformed human B cells, with HEF1 being the predominant substrate. Ligation of the B cell antigen receptor (BCR) also induced tyrosine phosphorylation of predominantly HEF1, and similar to integrins, BCR-mediated HEF1 phosphorylation was dependent upon an intact actin network. We further showed that Cas and HEF1 complexed in vivo with the related adhesion focal tyrosine kinase RAFTK, the adapter protein CrkL, and Lyn kinase, indicating that both molecules may play an important role in B cell signaling.
Background: The glycoprotein CD226 plays a key role in regulating immune cell function. Soluble CD226 (sCD226) is increased in sera of patients with several chronic inflammatory diseases but its levels in neuroinflammatory diseases such as multiple sclerosis (MS) are unknown. Objective: To investigate the presence and functional implications of sCD226 in persons with multiple sclerosis (pwMS) and other neurological diseases. Methods: The mechanisms of sCD226 production were first investigated by analyzing CD226 surface expression levels and supernatants of CD3/CD226-coactivated T cells. The role of sCD226 on dendritic cell maturation was evaluated. The concentration of sCD226 in the sera from healthy donors (HD), pwMS, neuromyelitis optica (NMO), and Alzheimer’s disease (AD) was measured. Results: CD3/CD226-costimulation induced CD226 shedding. Addition of sCD226 to dendritic cells during their maturation led to an increased production of the pro-inflammatory cytokine interleukin (IL)-23. We observed a significant increase in sCD226 in sera from pwMS and NMO compared to HD and AD. In MS, levels were increased in both relapsing–remitting multiple sclerosis (RRMS) and secondary-progressive multiple sclerosis (SPMS) compared to clinically isolated syndrome (CIS). Conclusion: Our data suggest that T-cell activation leads to release of sCD226 that could promote inflammation and raises the possibility of using sCD226 as a biomarker for neuroinflammation.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)