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The CC Chemokine Monocyte Chemotactic Peptide-1 Activates both the Class I p85/p110 Phosphatidylinositol 3-Kinase and the Class II PI3K-C2α*

Open AccessPublished:October 02, 1998DOI:https://doi.org/10.1074/jbc.273.40.25987
      The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 ± 1.88 nm,n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 ± 4.2 nm, n= 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2α) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2α activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2α rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2α. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2α enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences.
      MCP-1
      monocyte chemotactic peptide-1
      HPLC
      high performance liquid chromatography
      mAb
      monoclonal antibody
      PI
      phosphatidylinositol
      PI 3-P
      phosphatidylinositol 3-monophosphate
      PI 3
      4-P2, phosphatidylinositol 3,4-bisphosphate
      PI 3
      4,5-P3, phosphatidylinositol 3,4,5-trisphosphate
      PTK
      protein-tyrosine kinase
      RANTES
      regulated on activation normal T cell expressed and secreted
      CCR2
      CC chemokine receptor 2.
      Chemokines are a rapidly growing superfamily of 8–10-kDa peptides that selectively attract and activate leukocyte populations (
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ,
      • Rollins B.J.
      ). Monocyte chemotactic peptide-1 (MCP-1)1 (
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ) is a member of the CC chemokine family (
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ,
      • Rollins B.J.
      ), is a potent inducer of monocyte and CD45RO+ lymphocyte chemotaxis (
      • Furutani Y.
      • Nomura M.
      • Notake Y.
      • Oyamada T.
      • Fukui N.
      • Yamada C.G.
      • Oppenheim J.J.
      • Matsushima K.
      ,
      • Carr M.W.
      • Roth S.J.
      • Luther E.
      • Rose S.S.
      • Springer T.A.
      ), and also activates host defense mechanisms such as superoxide release (
      • Zachariae C.O.C.
      • Anderson A.O.
      • Thompson H.L.
      • Appella E.
      • Mantovani A.
      • Oppenheim J.J.
      • Matsushima K.
      ). In vivo studies suggest that MCP-1 recruits monocytes to sites of inflammation in a variety of pathological conditions including atherosclerosis (
      • Nelken N.A.
      • Coughlin S.R.
      • Gordon D.
      • Wilcox J.N.
      ) and rheumatoid arthritis (
      • Koch A.E.
      • Kunkel S.L.
      • Harlow L.A.
      • Johnson B.
      • Evanoff H.L.
      • Haines G.K.
      • Burdick M.D.
      • Pope R.M.
      • Streiter R.M.
      ) as well as pulmonary fibrosis and granulomatous lung disease (
      • Chensue S.W.
      • Warmington K.S.
      • Ruth J.H.
      • Sanghi P.S.
      • Lincoln P.
      • Kunkel S.L.
      ). MCP-1 has also been demonstrated to augment cytotoxic lymphocyte and natural killer cell activity in vitro, suggesting a novel role for chemokines as costimulators of T cell activation (
      • Taub D.D.
      • Ortaldo J.R.
      • Turcovski-Corrales S.M.
      • Key M.L.
      • Longo D.L.
      • Murphy W.J.
      ). Support for MCP-1's importance in the physiology of inflammation comes from demonstrations in transgenic mice that it functions as a monocyte chemoattractantin vivo (
      • Fuentes M.E.
      • Durham S.K.
      • Swerdel M.R.
      • Lewin A.C.
      • Barton D.S.
      • Megill J.R.
      • Bravo R.
      • Lira S.A.
      ,
      • Grewal I.S.
      • Rutledge B.J.
      • Fiorillo J.A.
      • Gu L.
      • Gladue R.P.
      • Flavell R.A.
      • Rollins B.J.
      ,
      • Gunn M.D.
      • Nelken N.A.
      • Liao X.
      • Williams L.T.
      ). Moreover, abnormalities in monocyte recruitment and cytokine expression are observed in MCP-1-deficient mice (
      • Lu B.
      • Rutledge B.J.
      • Gu L.
      • Fiorillo J.
      • Lukacs N.W.
      • Kunkel S.L.
      • North R.
      • Gerard C.
      • Rollins B.J.
      ).
      The effects of chemokines are mediated by a family of closely related G protein-coupled receptors (
      • Baggiolini M.
      • Dewald B.
      • Moser B.
      ). MCP-1 binds to CC chemokine receptor 2 (CCR2), which exists in A or B forms that arise via alternative splicing of the carboxyl-terminal tail (
      • Charo I.F.
      • Myers S.J.
      • Herman A.
      • Franci C.
      • Connolly A.J.
      • Coughlin S.R.
      ). CCR2 is activated by multiple agonists, including MCP-2 (
      • Gong X.
      • Gong W.
      • Kuhns D.B.
      • Ben-Baruch A.
      • Howard O.M.Z.
      • Wang J.M.
      ), MCP-3 (
      • Franci C.
      • Wong L.M.
      • Van Damme J.
      • Proost P.
      • Charo I.F.
      ,
      • Combadiere C.
      • Ahuja S.K.
      • Van Damme J.
      • Tiffany H.L.
      • Gao J.
      • Murphy P.M.
      ), MCP-4 (
      • Garcia-Zepeda E.A.
      • Combadiere C.
      • Rothenberg M.E.
      • Sarafi M.N.
      • Lavigne F.
      • Hamid Q.
      • Murphy P.M.
      • Luster A.D.
      ,
      • Stellato C.P.
      • Collins P.
      • Ponath P.D.
      • Soler D.
      • Newman W.
      • La Rosa G.
      • Li H.
      • White J.
      • Schwiebert L.M.
      • Bickel C.
      • Liu M.
      • Bochner B.S.
      • Williams T.J.
      • Schleimer R.P.
      ), and MCP-5 (
      • Sarafi M.N.
      • Garcia-Zepeda E.A.
      • Maclean J.A.
      • Charo I.F.
      • Luster A.D.
      ). In addition, only chemokines of the MCP family (MCP-1, -2, -3, -4, and -5) appear to activate CCR2, although CCR2 agonists can also bind and activate other receptors, since MCP-3 activates CCR1 (
      • Franci C.
      • Wong L.M.
      • Van Damme J.
      • Proost P.
      • Charo I.F.
      ,
      • Combadiere C.
      • Ahuja S.K.
      • Van Damme J.
      • Tiffany H.L.
      • Gao J.
      • Murphy P.M.
      ) and MCP-3 and MCP-4 activate CCR3 (
      • Garcia-Zepeda E.A.
      • Combadiere C.
      • Rothenberg M.E.
      • Sarafi M.N.
      • Lavigne F.
      • Hamid Q.
      • Murphy P.M.
      • Luster A.D.
      ,
      • Stellato C.P.
      • Collins P.
      • Ponath P.D.
      • Soler D.
      • Newman W.
      • La Rosa G.
      • Li H.
      • White J.
      • Schwiebert L.M.
      • Bickel C.
      • Liu M.
      • Bochner B.S.
      • Williams T.J.
      • Schleimer R.P.
      ). Studies with CCR2−/− mice have recently revealed, however, that MCP-1 initiates cellular responses primarily through binding to CCR2 (
      • Boring L.
      • Gosling J.
      • Chensue S.W.
      • Kunkel S.L.
      • Farese R.V.
      • Broxmeyer H.E.
      • Charo I.F.
      ). Analysis of the signal transduction pathways activated by MCP-1 has revealed pertussis toxin-sensitive phospholipase C activation (
      • Kuang Y.
      • Wu Y.
      • Jiang H.
      • Wu D.
      ), elevation of intracellular calcium (
      • Charo I.F.
      • Myers S.J.
      • Herman A.
      • Franci C.
      • Connolly A.J.
      • Coughlin S.R.
      ,
      • Sozzani S.
      • Molino M.
      • Locati M.
      • Luini W.
      • Cerletti C.
      • Vecchi A.
      • Mantovani A.
      ), and inhibition of adenyl cyclase (
      • Myers S.J.
      • Wong L.M.
      • Charo I.F.
      ). Interestingly, both CCR2A and CCR2B can both couple to the Gi-Gβγ-PLCβ2 pathway, but these receptors demonstrate an interesting specificity in their coupling to the α-subunits of the Gq class. Hence, CCR2B couples to both Gα16 and Gα14, whereas CCR2A cannot couple to either Gα14 or Gα16 (
      • Kuang Y.
      • Wu Y.
      • Jiang H.
      • Wu D.
      ). Other signaling events downstream of MCP-1 remain relatively poorly defined. In this respect, it is interesting to note that the related CC chemokine RANTES has been demonstrated to stimulate the tyrosine phosphorylation of a number of proteins in a T-cell clone (
      • Bacon K.B.
      • Szabo M.C.
      • Yssel H.
      • Bolen J.B.
      • Schall T.J.
      ,
      • Bacon K.B.
      • Premack B.A.
      • Gardner P.
      • Schall T.J.
      ) and to activate the protein-tyrosine kinase (PTK)/src homology domain 2-coupled phosphatidylinositol (PI) 3-kinase (
      • Turner L.
      • Ward S.G.
      • Westwick J.
      ), a member of the class 1A family of the phosphatidylinositol 3-kinases (
      • Vanhaesebroeck B.
      • Leevers S.A.
      • Panayotou G.
      • Waterfield M.D.
      ).
      The prototypical class 1A PI 3-kinase consists of an 85-kDa regulatory subunit responsible for protein-protein interactions via protein tyrosine phosphate-binding src homology domains and a catalytic 110-kDa subunit (
      • Vanhaesebroeck B.
      • Leevers S.A.
      • Panayotou G.
      • Waterfield M.D.
      ). PI 3-kinase is now regarded as an important intracellular signal that is upstream of a variety of responses including insulin-stimulated glucose uptake (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ), membrane ruffling (
      • Wennstrom S.
      • Hawkins P.T.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.R.
      ), superoxide production (
      • Arcaro A.
      • Wymann M.P.
      ), activation of p70 S6 kinase (
      • Chung J.
      • Grammer T.
      • Lemon C.
      • Kazlauskas A.
      • Blenis J.
      ), and activation of Akt/protein kinase B (
      • Burgering B.T.
      • Coffer P.J.
      ). A G protein-coupled PI 3-kinase, namely PI 3-kinase γ has also been identified (
      • Stephens L.R.
      • Eguinoa A.
      • Erdjument-Bromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.T.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ). PI 3-kinase γ is, to date, the only characterized member of the class 1B G protein-coupled PI 3-kinase family and consists of a unique 101-kDa regulatory subunit and a distinct 110-kDa catalytic subunit termed p110γ (
      • Vanhaesebroeck B.
      • Leevers S.A.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Burgering B.T.
      • Coffer P.J.
      ,
      • Stephens L.R.
      • Eguinoa A.
      • Erdjument-Bromage H.
      • Lui M.
      • Cooke F.
      • Coadwell J.
      • Smrcka A.S.
      • Thelen M.
      • Cadwallader K.
      • Tempst P.
      • Hawkins P.T.
      ). Nevertheless, there is some evidence that G protein-coupled receptors, such as fMLP receptors, are able to activate the p85/p110 PI 3-kinase (
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ). In this respect, the p85/p110 heterodimer has been demonstrated to be synergistically activated by the βγ subunits of G proteins and by phosphotyrosyl peptide (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ). The class I PI 3-kinases can potentially generate three lipid products, namely phosphatidylinositol 3-monophosphate (PI 3-P), phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2), and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3), which are collectively known as D-3 phosphatidylinositol lipids (reviewed in Refs.
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      and
      • Toker A.
      • Cantley L.C.
      ). To date, PI 3,4-P2 and PI 3,4,5-P3 are regarded as signaling molecules, whereas PI 3-P is thought to regulate membrane trafficking (
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Toker A.
      • Cantley L.C.
      ). The PI 3-kinase family is completed by the class II C2 domain-containing PI 3-kinases and the class III PtdIns-specific 3-kinases (
      • Vanhaesebroeck B.
      • Leevers S.A.
      • Panayotou G.
      • Waterfield M.D.
      ). The 190-kDa PI3K-C2α is a novel human member of the class II PI 3-kinase family that, in contrast to members of the other PI 3-kinase classes, is refractory to inhibition by the PI 3-kinase inhibitors wortmannin and LY294002 (
      • Domin J.
      • Pages F.
      • Volinia S.
      • Rittenhouse S.E.
      • Zvelebil M.J.
      • Stein R.C.
      • Waterfield M.D.
      ). In addition, PI3K-C2α utilizes predominantly PI and phosphatidylinositol 4-monophosphate as substrates in vitro, but when presented with phosphatidylserine it can also phosphorylate PI 4,5-bisphosphate (
      • Domin J.
      • Pages F.
      • Volinia S.
      • Rittenhouse S.E.
      • Zvelebil M.J.
      • Stein R.C.
      • Waterfield M.D.
      ).
      Given the functional role of MCP-1 on superoxide generation and chemotaxis (
      • Carr M.W.
      • Roth S.J.
      • Luther E.
      • Rose S.S.
      • Springer T.A.
      ,
      • Zachariae C.O.C.
      • Anderson A.O.
      • Thompson H.L.
      • Appella E.
      • Mantovani A.
      • Oppenheim J.J.
      • Matsushima K.
      ), it is interesting to note that the PI 3-kinase inhibitor wortmannin inhibits fMLP-stimulated superoxide release (
      • Arcaro A.
      • Wymann M.P.
      ), interleukin-8-stimulated neutrophil chemotaxis (
      • Knall C.
      • Worthen G.S.
      • Johnson G.L.
      ), and RANTES-stimulated chemotaxis of T-lymphocyte (
      • Turner L.
      • Ward S.G.
      • Westwick J.
      ). In this study, therefore, we have investigated the possible involvement of PI 3-kinase(s) in MCP-1 signal transduction and chemotaxis using the THP-1 monocytic cell line.

      DISCUSSION

      This study has demonstrated that MCP-1 induces a concentration- and time-dependent accumulation of both PI 3,4-P2 and PI 3,4,5-P3 in THP-1 cells. This MCP-1-stimulated generation of D-3 phosphatidylinositol lipids was not affected by pretreatment of the cells with wortmannin but was greatly reduced by pertussis toxin. Further characterization of the PI 3-kinase family members that act downstream of the MCP-1-stimulated receptor, demonstrated that MCP-1 activates the p85/p110 heterodimeric PI 3-kinase, but this activation was very sensitive to wortmannin and insensitive to pertussis toxin. Concentrations of wortmannin sufficient to inhibit p85/p110 lipid kinase activity also inhibited MCP-1-stimulated THP-1 cell chemotaxis. Investigation of the MCP-1-induced tyrosine phosphorylation of various proteins demonstrated that three proteins were rapidly and transiently phosphorylated by MCP-1 in THP-1 cells, although a fourth tyrosine phosphoprotein was found to co-precipitate with the p85 subunit of PI 3-kinase. Moreover, following the relative insensitivity of the MCP-1-induced PI 3,4-P2 and PI 3,4,5-P3 accumulation to wortmannin, we demonstrated the MCP-1-induced activation of the novel PI3K-C2α in a wortmannin-resistant, but pertussis toxin-sensitive manner. These results provide the first demonstration that MCP-1 stimulates D-3 phosphatidylinositol lipid production and that MCP-1 activates the p85/p110 PI 3-kinase, which correlates with activation of PTK(s). Furthermore, our data provide the first evidence for agonist-induced activation of PI3K-C2α.
      MCP-1 appears to have differential effects on the accumulation of PI 3,4-P2 and PI 3,4,5-P3. For instance, MCP-1 stimulates a rapid and transient increase in PI 3,4,5-P3compared with the slower, sustained increase in PI 3,4-P2. This may be explained by the concomitant activation of a PI 3,4,5-P3 5-phosphatase, which has been identified in other systems (
      • Damen J.E.
      • Liu L.
      • Rosten P.
      • Humphries R.K.
      • Jefferson A.B.
      • Majerus P.W.
      • Krystal G.
      ,
      • Ono M.
      • Bolland S.
      • Tempst P.
      • Ravetch J.V.
      ,
      • Lioubin M.N.
      • Algate P.A.
      • Tsai S.
      • Carlberg K.
      • Aebersold R.
      • Rohrschneider L.R.
      ) and which converts PI 3,4,5-P3 to PI 3,4-P2. The elevation of D-3 phosphatidylinositol lipids observed in response to MCP-1 may be the result of activation of more than one PI 3-kinase (e.g. the p85/p110 PI 3-kinase, PI 3-kinase γ, and/or other classes of PI 3-kinase such as PI3K-C2α). However, the use of pharmacological inhibitors revealed some interesting and important points concerning the in vivoaccumulation of D-3 phosphatidylinositol lipids stimulated by MCP-1. First, the accumulation of D-3 phosphatidylinositol lipids in THP-1 cells stimulated by MCP-1 could be completely inhibited by pretreatment with pertussis toxin, strongly indicating that D-3 phosphatidylinositol lipid accumulation occurs via a pertussis toxin-sensitive G-protein-coupled PI 3-kinase. Crucially however, the lack of effect of wortmannin on the MCP-1-induced D-3 phosphatidylinositol lipid generation indicated that it is likely that MCP-1 activates a PI 3-kinase distinct from PI 3-kinase γ, since PI 3-kinase γ is known to be inhibited by the concentrations of wortmannin used in this study (
      • Vanhaesebroeck B.
      • Leevers S.A.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ). Second, although MCP-1 activates a wortmannin-sensitive (but pertussis toxin-resistant) p85/p110 heterodimer in vitro, the slower kinetics of p85/p110 activation and the lack of effect of wortmannin on D-3 phosphatidylinositol lipid generation in vivo suggest that the formation of D-3 phosphoinositide lipids resulting from p85/p110 activation does not significantly contribute to the overall detectable D-3 phosphatidylinositol lipid pool. Nevertheless, activation of the p85/p110 heterodimer appears to be functionally relevant, since wortmannin inhibits MCP-1 stimulated THP-1 chemotaxis. Our data are similar to previous findings of other groups who have reported that concanavalin A-stimulated generation of D-3 phosphatidylinositol lipids in THP-1 cells is completely inhibited by pertussis toxin, while the increased PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates is unaffected by pertussis toxin (
      • Matsuo T.
      • Hazeki K.
      • Hazeki O.
      • Katada T.
      • Ui M.
      ).
      Our surprising observations using wortmannin and pertussis toxin led us to look at the effects of MCP-1 on the novel PI3K-C2α, which has recently been identified as displaying reduced sensitivity to wortmannin (
      • Domin J.
      • Pages F.
      • Volinia S.
      • Rittenhouse S.E.
      • Zvelebil M.J.
      • Stein R.C.
      • Waterfield M.D.
      ). MCP-1 stimulation of THP-1 cells did activate PI3K-C2α, and this activation was resistant to pretreatment with wortmannin but inhibited by pertussis toxin. The effects of wortmannin and pertussis toxin on PI3K-C2α in vitro mirror the effects of these inhibitors on the MCP-1-stimulated increases in D-3 phosphatidylinositol lipid generation. Thus, the pertussis toxin-sensitive MCP-1-induced activation of PI3K-C2α may account for the detectable changes in PI 3,4-P2 and PI 3,4,5-P3 observed in vivo. From analogy with what is known about receptor coupling to PI 3-kinase γ, there are at least two putative mechanisms by which MCP-1 can activate PI3K-C2α in a pertussis-sensitive manner: (i) direct interaction of Gβγ subunits with PI3K-C2α, as is known to occur for PI 3-kinase γ activation (
      • Leopoldt D.
      • Hanck T.
      • Exner T.
      • Maier U.
      • Wetzker R.
      • Nurnberg B.
      ); (ii) Gβγ subunits may activate PI3K-C2α via PTK(s) given that there are several lines of evidence to support involvement of PTKs in cell signaling mediated by pertussis toxin-sensitive G proteins (
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Ptasznik A.
      • Traynor-Kaplan A.
      • Bokoch G.M.
      ,
      • Hawes B.E.
      • Luttrell L.M.
      • van Biesen T.
      • Lefkowitz R.J.
      ,
      • Lopez-Ilasca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ). In this respect, it is interesting to note that the MCP-1-stimulated tyrosine phosphorylation of proteins is inhibited by pertussis toxin pretreatment. However, until effective PI3K-C2α mutants and or pharmacological inhibitors of PI3K-C2α are available, the precise functional role of this PI3K-C2α activation in mediating MCP-1-induced responses cannot be determined. Our data do not, however, exclude the possibility that MCP-1 activates a unique wortmannin-resistant, class 1B G protein-coupled PI 3-kinase that is distinct from p110γ, which might also contribute (along with PI3K-C2α) to the observed accumulation of PI 3,4-P2 and PI 3,4,5-P3.
      Coupling of receptors to the class 1A p85/p110 PI 3-kinase is known to require interaction of src homology 2 domains within the p85 regulatory subunit with specific phosphotyrosine-containing binding motifs (pYXXM; where pY represents phosphotyrosine) located in several growth factor receptors or adaptor molecules such as the insulin receptor substrate-1 (
      • Ward S.G.
      • June C.H.
      • Olive D.
      ,
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.
      ). The mechanisms by which the known G protein-coupled MCP-1 receptor couples to class 1A PI 3-kinase are unclear, since there is no recognized consensus binding motif for the p85 srchomology 2 domains contained within the CCR2 sequence. Since pertussis toxin had no effect on the MCP-1-induced activation of the p85/p110 PI 3-kinase, the coupling of the MCP-1 receptor to a class 1API 3-kinase would have to involve pertussis toxin-insensitive G-proteins. In this respect, it is interesting to note that CCR2A and CCR2B are differentially coupled to the pertussis toxin-insensitive α-subunits of the Gq class of G proteins (
      • Kuang Y.
      • Wu Y.
      • Jiang H.
      • Wu D.
      ). Given the tyrosine phosphorylation of proteins after MCP-1 stimulation, it appears that MCP-1 stimulation of CCR2 can result in activation of PTK(s). Indeed, other G-protein-coupled receptors have also been shown to stimulate PTKs such as bombesin and vasopressin (
      • Zachary I.
      • Gil J.
      • Lehmann W.
      • Sinnett-Smith L.
      • Rozengurt E.
      ). Similarly, additional reports indicate that MCP-1 and the related CC chemokine RANTES can induce the protein tyrosine phosphorylation of a number of substrates (
      • Bacon K.B.
      • Szabo M.C.
      • Yssel H.
      • Bolen J.B.
      • Schall T.J.
      ,
      • Bacon K.B.
      • Premack B.A.
      • Gardner P.
      • Schall T.J.
      ). Furthermore, MCP-1-induced tyrosine phosphorylation of p42/p44 mitogen-activated protein kinases in a murine T-cell hybridoma expressing human MCP-1 receptors has previously been reported (
      • Dubois P.M.
      • Palmer D.
      • Webb M.L.
      • Ledbetter J.A.
      • Shapiro R.A.
      ). While we were unable to detect tyrosine phosphorylation of comparable proteins in THP-1 cells, MCP-1 treatment did stimulate protein tyrosine phosphorylation of 50-, 80-, and 120-kDa proteins. Our findings that MCP-1-induced tyrosine phosphorylation of these proteins was inhibited by pertussis toxin demonstrates that this is downstream of pertussis toxin-sensitive Gi and/or Go G proteins. In contrast, the use of wortmannin indicates that PI 3-kinase activation is not a requirement for MCP-1-stimulated tyrosine phosphorylation. In addition to the above mentioned tyrosine-phosphorylated proteins, a 55-kDa phosphoprotein co-associates with p85 immunoprecipitates upon MCP-1 stimulation. The 55-kDa phosphoprotein was tyrosine-phosphorylated with slower kinetics than the other three proteins that were detected in anti-phosphotyrosine immunoprecipitates. This delayed appearance of the p85-associated 55-kDa phosphoprotein generally correlated with the kinetics of p85/p110 PI 3-kinase activation, which were slightly slower than the elevation of D-3 phosphatidylinositol lipids and PI3K-C2α activation. Therefore, it is possible that these tyrosine phosphoproteins, in particular the 55-kDa protein, could be involved in the coupling of the MCP-1 receptor to the p85/p110 PI 3-kinase. In this respect, activation of p85/p110 PI 3-kinase following binding of tyrosine phosphopeptides (
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ,
      • Backer J.M.
      • Myers M.G.
      • Shoelson S.
      • Chin D.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ) and synergistic activation of p85/p110 by tyrosine-phosphorylated peptides and βγ subunits of GTP binding proteins have both been reported (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ,
      • Okada T.
      • Hazeki O.
      • Ui M.
      • Katada T.
      ).
      This investigation has demonstrated that MCP-1 activates at least two independent PI 3-kinase isoforms that may differentially regulate monocyte functions. For instance, although both the p85/p110 PI 3-kinase and PI 3-kinase γ have been shown to be activated by thrombin, studies with wortmannin indicated that only the p85/p110 PI 3-kinase complex was involved in regulating the conversion of the platelet integrin αIIbβ3 into a fibrinogen binding form required for platelet aggregation (
      • Zhang J.
      • Zhang J.
      • Shattil S.J.
      • Cunningham M.C.
      • Rittenhouse S.E.
      ). Indeed, it has previously been proposed that phosphotyrosine-linked activation of PI 3-kinase is responsible for phagocytosis, whereas G protein-mediated activation of PI 3-kinase gives rise to the respiratory burst (
      • Matsuo T.
      • Hazeki K.
      • Hazeki O.
      • Katada T.
      • Ui M.
      ). PI 3-kinase activation has been implicated in a variety of cellular responses such as adhesion molecule up-regulation (
      • Shimizu Y.
      • Hunt S.
      ), superoxide release (
      • Chung J.
      • Grammer T.
      • Lemon C.
      • Kazlauskas A.
      • Blenis J.
      ), and chemotaxis (
      • Turner L.
      • Ward S.G.
      • Westwick J.
      ,
      • Knall C.
      • Worthen G.S.
      • Johnson G.L.
      ). The MCP-1-induced activation of a wortmannin-sensitive PI 3-kinase appears to be an important signal required for MCP-1-stimulated chemotaxis. Our data indicate that the p85/p110 heterodimer is activated by MCP-1 and may be responsible for mediating the MCP-1 effects on chemotaxis. However, until a suitable inhibitor of PI3K-C2α becomes available, the role of this class II PI 3-kinase in MCP-1-induced monocyte functional responses will remain elusive.

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