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Hck Enhances the Adherence of Lipopolysaccharide-stimulated Macrophages via Cbl and Phosphatidylinositol 3-Kinase*

  • Glen Scholz
    Correspondence
    To whom correspondence should be addressed. Tel.: 61-3-9341-3155; Fax: 61-3-9341-3191
    Affiliations
    Molecular Biology Laboratory, Ludwig Institute for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia
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  • Kellie Cartledge
    Affiliations
    Molecular Biology Laboratory, Ludwig Institute for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia
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  • Ashley R. Dunn
    Affiliations
    Molecular Biology Laboratory, Ludwig Institute for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia
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  • Author Footnotes
    * This work was supported in part by a grant from the National Health and Medical Research Council (to G. S. and A. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:May 12, 2000DOI:https://doi.org/10.1074/jbc.275.19.14615
      Src family tyrosine kinases have previously been proposed to mediate some of the biological effects of lipopolysaccharide on macrophages. Accordingly, we have sought to identify substrates of Src family kinases in lipopolysaccharide-stimulated macrophages. Stimulation of Bac1.2F5 macrophage cells with lipopolysaccharide was found to induce gradual and persistent tyrosine phosphorylation of Cbl in an Src family kinase-dependent manner. Immunoprecipitation experiments revealed that Cbl associates with Hck in Bac1.2F5 cells, while expression of an activated form of Hck in Bac1.2F5 cells induces tyrosine phosphorylation of Cbl in the absence of lipopolysaccharide stimulation. The Src homology 3 domain of Hck can directly bind Cbl, and this interaction is important for phosphorylation of Cbl. Association of the p85 subunit of phosphatidylinositol (PI) 3-kinase with Cbl is enhanced following lipopolysaccharide stimulation of Bac1.2F5 cells, and transient expression experiments indicate that phosphorylation of Cbl by Hck can facilitate the association of p85 with Cbl. Lipopolysaccharide treatment also stimulates the partial translocation of Hck to the cytoskeleton of Bac1.2F5 cells. Notably, lipopolysaccharide enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. Thus, we postulate that Hck enhances the adherence of lipopolysaccharide-stimulated macrophages, at least in part, via Cbl and PI 3-kinase.
      LPS
      lipopolysaccharide
      PI
      phosphatidylinositol
      PBS
      phosphate-buffered saline
      GST
      glutathione S-transferase
      SH2 and SH3
      Src homology 2 and 3, respectively
      Pipes
      1,4-piperazinediethanesulfonic acid
      Macrophages play a critical role in the host response to inflammation and bacterial infection (
      • Auger M.J.
      • Ross J.A.
      ). During these processes, macrophages are primarily involved in the phagocytosis of bacteria and host cell debris, antigen processing and presentation, and the secretion of reactive nitrogen intermediates and inflammatory cytokines (e.g. tumor necrosis factor α, interleukin-1, and interleukin-6) (
      • Auger M.J.
      • Ross J.A.
      ). In vitro exposure of macrophages to lipopolysaccharide (LPS),1 a major component of the outer wall of Gram-negative bacteria, mimics many of the effects bacteria have on macrophages in vivo, namely inducing the secretion of reactive nitrites and inflammatory cytokines and enhancing their tumoricidal activity (
      • Auger M.J.
      • Ross J.A.
      ).
      The activation of macrophages by LPS is mediated by the binding of LPS to CD14, a glycosylphosphatidylinositol-anchored protein found on the surface of monocytes/macrophages and neutrophils (
      • Wright S.D.
      • Ramos R.A.
      • Tobias P.S.
      • Ulevitch R.J.
      • Mathison J.C.
      ). Binding of LPS to CD14 is enhanced by LBP, an LPS-binding protein found in serum (
      • Ulevitch R.J.
      • Tobias P.S.
      ). Since CD14 lacks an intracellular domain, it has previously been unclear how CD14 transduces signals across the plasma membrane in response to the binding of LPS. Recent studies suggest that members of the Toll-like receptor family, and in particular Toll-like receptor 4, may serve as cell surface co-receptors with CD14 to mediate transmembrane signal transduction (
      • Yang R.B.
      • Mark M.R.
      • Gray A.
      • Huang A.
      • Xie M.H.
      • Zhang M.
      • Goddard A.
      • Wood W.I.
      • Gurney A.L.
      • Godowski P.J.
      ,
      • Kirschning C.J.
      • Wesche H.
      • Merrill-Ayres T.
      • Rothe M.
      ,
      • Heine H.
      • Kirschning C.J.
      • Lien E.
      • Monks B.G.
      • Rothe M.
      • Golenbock D.T.
      ,
      • Yang R.B.
      • Mark M.R.
      • Gurney A.L.
      • Godowski P.J.
      ,
      • Poltorak A.
      • He X.
      • Smirnova I.
      • Liu M.Y.
      • Huffel C.V.
      • Du X.
      • Birdwell D.
      • Alejos E.
      • Silva M.
      • Galanos C.
      • Freudenberg M.
      • Ricciardi-Castagnoli P.
      • Layton B.
      • Beutler B.
      ,
      • Chow J.C.
      • Young D.W.
      • Golenbock D.T.
      • Christ W.J.
      • Gusovsky F.
      ,
      • Qureshi S.T.
      • Lariviere L.
      • Leveque G.
      • Clermont S.
      • Moore K.J.
      • Gros P.
      • Malo D.
      ,
      • Hoshino K.
      • Takeuchi O.
      • Kawai T.
      • Sanjo H.
      • Ogawa T.
      • Takeda Y.
      • Takeda K.
      • Akira S.
      ). Members of the Toll-like receptor family are structurally characterized by an extracellular domain containing leucine-rich repeats, a transmembrane domain, and an intracellular domain with sequence homology to the intracellular domain of the interleukin-1 receptor (
      • Rock F.L.
      • Hardiman G.
      • Timans J.C.
      • Kastelein R.A.
      • Bazan J.F.
      ).
      LPS stimulation of macrophages leads to the activation of a variety of proteins involved in signal transduction, including protein kinase C (
      • Novotney M.
      • Chang Z.L.
      • Uchiyama H.
      • Suzuki T.
      ,
      • Shapira L.
      • Takashiba S.
      • Champagne C.
      • Amar S.
      • Van Dyke T.E.
      ), Raf (
      • Reimann T.
      • Buscher D.
      • Hipskind R.
      • Krautwald S.
      • Lohmann-Matthes M.L.
      • Baccarini M.
      ), mitogen-activated protein kinase (
      • Reimann T.
      • Buscher D.
      • Hipskind R.
      • Krautwald S.
      • Lohmann-Matthes M.L.
      • Baccarini M.
      ,
      • Weinstein S.L.
      • Sanghera J.S.
      • Lemke K.
      • DeFranco A.L.
      • Pelech S.L.
      ), p38 stress-activated protein kinase (
      • Han J.
      • Lee J.D.
      • Bibbs L.
      • Ulevitch R.J.
      ), Jun kinase (
      • Hambleton J.
      • Weinstein S.L.
      • Lem L.
      • DeFranco A.L.
      ), and ceramide-activated protein kinase (
      • Joseph C.K.
      • Wright S.D.
      • Bornmann W.G.
      • Randolph J.T.
      • Kumar E.R.
      • Bittman R.
      • Liu J.
      • Kolesnick R.N.
      ). However, it is still unclear how the activation of these various signal-transducing proteins mediates the variety of biological responses of macrophages to LPS. Significantly, both in vitro and in vivo studies with tyrosine kinase inhibitors have revealed that the activation of tyrosine kinases is necessary for a number of the biological responses of macrophages to LPS (e.g. tumoricidal activation) (
      • Dong Z.
      • O'Brian C.A.
      • Fidler I.J.
      ,
      • Geng Y.
      • Zhang B.
      • Lotz M.
      ,
      • Novogrodsky A.
      • Vanichkin A.
      • Patya M.
      • Gazit A.
      • Osherov N.
      • Levitzki A.
      ).
      The Src family tyrosine kinases Hck, Lyn, and Fgr have all been implicated in playing a role in the biological response of macrophages to LPS. Stimulation of monocytes/macrophages with LPS induces a rapid increase in the specific kinase activity of Hck, Lyn, and Fgr and physical association of Lyn with CD14 (
      • Stefanova I.
      • Corcoran M.L.
      • Horak E.M.
      • Wahl L.M.
      • Bolen J.B.
      • Horak I.D.
      ,
      • English B.K.
      • Ihle J.N.
      • Myracle A.
      • Yi T.
      ). Moreover, enforced expression of an activated form of Hck in Bac1.2F5 macrophage cells augments tumor necrosis factor α production in response to LPS stimulation (
      • English B.K.
      • Ihle J.N.
      • Myracle A.
      • Yi T.
      ). Chronic exposure of bone marrow-derived macrophages to LPS induces an increase in the expression of both Hck and Lyn (
      • Boulet I.
      • Ralph S.
      • Stanley E.
      • Lock P.
      • Dunn A.R.
      • Green S.P.
      • Phillips W.A.
      ). Analysis of the promoter region of the hck gene has facilitated definition of an element that confers LPS responsiveness (
      • Lock P.
      • Stanley E.
      • Holtzman D.A.
      • Dunn A.R.
      ). Although these observations suggest that Src family kinases play a role in the response of macrophages to LPS, and hence in the response of macrophages to bacterial infection, the critical substrates of Src family kinases that mediate the various biological responses of macrophage to LPS have yet to be identified.
      In the present study we have sought to identify proteins that are phosphorylated by Src family kinases following LPS stimulation of the Bac1.2F5 macrophage cell line. We show that Cbl is a substrate of Hck in LPS-stimulated Bac1.2F5 cells. Further, we show that the phosphorylation of Cbl facilitates the physical association of the p85 subunit of PI 3-kinase with Cbl. Notably, LPS stimulation enhances the adherence of Bac1.2F5 cells, an effect that is dependent on the activity of Src family kinases and PI 3-kinase. On the basis of these findings, we postulate that Hck, at least in part, enhances the adherence of LPS-stimulated macrophages via Cbl and PI 3-kinase.

      DISCUSSION

      In the present study, we sought to identify proteins that are phosphorylated by Src family kinases following LPS stimulation of Bac1.2F5 cells, since these proteins may play a critical role in the biological response of macrophages to LPS. We have shown that one of the proteins that became tyrosine-phosphorylated in an Src family kinase-dependent manner following LPS stimulation of Bac1.2F5 cells was Cbl. Tyrosine phosphorylation of Cbl was found to be both gradual and persistent. Maximal tyrosine phosphorylation of Cbl did not occur until 15–30 min after LPS stimulation and remained elevated for at least 2 h. Such kinetics of tyrosine phosphorylation of Cbl contrast with the rapid and transient tyrosine phosphorylation of Cbl following colony-stimulating factor-1 stimulation of Bac1.2F5 cells (
      • Wang Y.
      • Yeung Y.G.
      • Langdon W.Y.
      • Stanley E.R.
      ) but are somewhat similar to those observed upon plating macrophages onto fibronectin-coated tissue culture dishes (
      • Ojaniemi M.
      • Martin S.S.
      • Dolfi F.
      • Olefsky J.M.
      • Vuori K.
      ). Significantly, we found that Cbl is physically associated with Hck in Bac1.2F5 cells and that enforced expression of a constitutively activated form of Hck in Bac1.2F5 cells induces tyrosine phosphorylation of Cbl in the absence of LPS stimulation. Additionally, Hck was shown to be capable of directly phosphorylating Cbl in vitro. Taken together, these findings are consistent with the notion that Hck directly mediates, at least in part, the phosphorylation of Cbl in LPS-stimulated Bac1.2F5 cells.
      By employing GST fusion proteins of Hck, we have been able to demonstrate that the association of Cbl with Hck is mediated by a direct interaction of Cbl with the SH3 domain of Hck. The interaction of Cbl with the SH3 domain of Hck is likely to be important for its subsequent phosphorylation, since an SH3 domain mutant of Hck499F was found to be 4–5-fold less efficient than Hck499F in phosphorylating Cbl in transiently transfected 293T cells. The reduced ability of the SH3 domain mutant to phosphorylate Cbl does not appear to be a consequence of the mutation negatively impacting on its specific activity, since its ability to phosphorylate other proteins (e.g. endogenous cellular proteins or co-transfected paxillin) was comparable with that of Hck499F. Since these experiments were performed in an overexpression system (i.e. 293T cells), the 4–5-fold lower phosphorylation of Cbl by the Hck499F SH3 domain mutant may actually underestimate the contribution of the SH3 domain of Hck to the phosphorylation of Cbl when the proteins are expressed at physiologically relevant levels (e.g. in Bac1.2F5 cells).
      Our finding that the SH3 domain of Hck is sufficient to bind Cbl contrasts with two previous reports describing interactions between Hck and Cbl (
      • Anderson S.M.
      • Burton E.A.
      • Koch B.L.
      ,
      • Howlett C.J.
      • Bisson S.A.
      • Resek M.E.
      • Tigley A.W.
      • Robbins S.M.
      ). The GST-SH3 domain fusion protein utilized in this study encompassed amino acids 72–140 of murine Hck, whereas the GST-SH3 domain fusion proteins employed in the previous studies encompassed amino acids 87–137 (
      • Anderson S.M.
      • Burton E.A.
      • Koch B.L.
      ,
      • Howlett C.J.
      • Bisson S.A.
      • Resek M.E.
      • Tigley A.W.
      • Robbins S.M.
      ). X-ray crystallographic and NMR studies, however, have revealed that the SH3 domain of Hck is formed by amino acids 80–135 (
      • Sicheri F.
      • Moarefi I.
      • Kuriyan J.
      ,
      • Horita D.A.
      • Baldisseri D.M.
      • Zhang W.
      • Altieri A.S.
      • Smithgall T.E.
      • Gmeiner W.H.
      • Byrd R.A.
      ). Thus, the fact that the GST fusion proteins employed in the prior studies lacked amino acids 80–86 may potentially explain the inability of those fusion proteins to bind Cbl. Our observation that the SH3 domain of Hck is capable of binding Cbl is consistent with previous reports that GST fusion proteins encompassing just the SH3 domain of other Src family kinases (e.g. Fyn, Lck, and Lyn) are capable of binding Cbl (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ).
      Even when tyrosine-phosphorylated, 2–3-fold more Cbl bound the SH3 domain of Hck when compared with that which bound the SH2 domain of Hck. Preferential binding of tyrosine-phosphorylated Cbl to the SH3 domain of Hck would have at least two important consequences. First, it may allow Hck to simultaneously bind another protein via its SH2 domain; second, the tyrosine residue(s) on Cbl that is phosphorylated by Hck may remain accessible to bind SH2 domain-containing proteins, thus potentially mediating the formation of a multiprotein-signaling complex. Tyrosine phosphorylation of Cbl has previously been reported to occur in response to a variety of stimuli, including cytokine stimulation (
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ,
      • Wang Y.
      • Yeung Y.G.
      • Langdon W.Y.
      • Stanley E.R.
      ,
      • Anderson S.M.
      • Burton E.A.
      • Koch B.L.
      ,
      • Barber D.L.
      • Mason J.M.
      • Fukazawa T.
      • Reedquist K.A.
      • Druker B.J.
      • Band H
      • D'Andrea A.D.
      ,
      • Ueno H.
      • Sasaki K.
      • Honda H.
      • Nakamoto T.
      • Yamagata T.
      • Miyagawa K.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ), activation of the T-cell and B-cell receptors (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ,
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Buday L.
      • Khwaja A.
      • Sipeki S.
      • Farago A.
      • Downward J.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Miyake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B
      • Schoelson S.
      • Cantley L.
      • Band H.
      ), cell adhesion (
      • Ojaniemi M.
      • Martin S.S.
      • Dolfi F.
      • Olefsky J.M.
      • Vuori K.
      ), and oncogenic transformation (
      • Andoniou C.E.
      • Thien C.B.
      • Langdon W.Y.
      ,
      • Ribon V.
      • Hubbell S.
      • Herrera R.
      • Saltiel A.R.
      ). Cbl is able to bind a number of SH3 domain-containing proteins involved in signal transduction (e.g. Grb2), and when tyrosine-phosphorylated bind SH2 domain-containing proteins (e.g. p85 subunit of PI 3-kinase) (
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ,
      • Anderson S.M.
      • Burton E.A.
      • Koch B.L.
      ,
      • Barber D.L.
      • Mason J.M.
      • Fukazawa T.
      • Reedquist K.A.
      • Druker B.J.
      • Band H
      • D'Andrea A.D.
      ,
      • Ueno H.
      • Sasaki K.
      • Honda H.
      • Nakamoto T.
      • Yamagata T.
      • Miyagawa K.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ,
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ,
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Buday L.
      • Khwaja A.
      • Sipeki S.
      • Farago A.
      • Downward J.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Miyake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B
      • Schoelson S.
      • Cantley L.
      • Band H.
      ,
      • Ojaniemi M.
      • Martin S.S.
      • Dolfi F.
      • Olefsky J.M.
      • Vuori K.
      ,
      • Ribon V.
      • Hubbell S.
      • Herrera R.
      • Saltiel A.R.
      ). These observations have led to the suggestion that Cbl may serve as a docking protein to facilitate assembly of multiprotein signaling complexes. Our finding that Hck phosphorylates Cbl in response to LPS stimulation of Bac1.2F5 cells suggests that Hck may mediate the formation of such a multiprotein-signaling complex in LPS-stimulated macrophages. Indeed, we have found that association of the p85 subunit of PI 3-kinase with Cbl is enhanced following LPS stimulation of Bac1.2F5 cells and that this association of p85 with Cbl is dependent on Src family kinase activity. Transient expression experiments in 293T cells have allowed us to demonstrate that phosphorylation of Cbl by Hck can facilitate the physical association of Cbl with p85. Moreover, we have been able to demonstrate that phosphorylation of Cbl on tyrosine 731 is necessary for its physical association with p85. Tyrosine 731 is found within the sequence CTYEAMYN, which conforms to the minimal consensus binding sequence of YXXM for the SH2 domains of p85 (
      • 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.C.
      ).
      What then is the biological significance of the LPS-induced tyrosine phosphorylation of Cbl by Hck? With regard to this question, it is worth noting that macrophages derived from mice simultaneously carrying null mutations at the hck, lyn, andfgr loci exhibit no discernible defects in terms of nitrite or inflammatory cytokine secretion when stimulated in vitrowith LPS (
      • Meng F.
      • Lowell C.A.
      ). Similarly, activation of the MAP kinase and JNK kinase signal transduction pathways by LPS appears normal inhck −/− lyn −/− fgr −/−triple mutant macrophages (
      • Meng F.
      • Lowell C.A.
      ). However, the ability of these macrophages to kill tumor cells in vitro is partially impaired (
      • Meng F.
      • Lowell C.A.
      ). Given that cell adhesion plays a role in tumor cell killing, this partial defect in macrophage function may be due to a cell adhesion defect. Indeed, Meng and Lowell (
      • Meng F.
      • Lowell C.A.
      ) have subsequently reported thathck −/− lyn −/− fgr −/−triple mutant macrophages have a major defect in β1integrin-mediated cell adhesion and spreading. In vivo this defect manifests itself in the form of reduced migration of macrophages into the peritoneum of mice injected with the inflammatory stimulus thioglycolate (
      • Meng F.
      • Lowell C.A.
      ). Taken together, the findings by Meng and Lowell (
      • Meng F.
      • Lowell C.A.
      ,
      • Meng F.
      • Lowell C.A.
      ) suggest that at the molecular level Hck, Lyn, and Fgr are not components of the signal transduction pathways that control nitrite and inflammatory cytokine secretion by macrophages, or if they are, another tyrosine kinase(s) can fulfill this function in their absence. However, these findings do suggest the three Src family kinases are indispensable components of the signal transduction pathways controlling macrophage adhesion/spreading and migration. Interestingly, a recent report has proposed that the activation state of Hck might serve as a “molecular switch” to regulate myelomonocytic motility and adherence in response to urokinase (
      • Chiaradonna F.
      • Fontana L.
      • Iavarone C.
      • Carriero M.V.
      • Scholz G.
      • Barone M.V.
      • Stoppelli M.P.
      ).
      Significantly, we have found that LPS stimulation increases the adherence of Bac1.2F5 cells. Moreover, the effect of LPS on Bac1.2F5 cell adhesion was found to be dependent on the activity of Src family kinases and PI 3-kinase and to correlate with the tyrosine phosphorylation status of Cbl. Accordingly, we propose that LPS may enhance the adherence of Bac1.2F5 cells via a Hck-Cbl-PI 3-kinase signal transduction pathway. Specifically, we propose that Hck directly phosphorylates Cbl on tyrosine 731 in response to LPS stimulation, thus facilitating the physical association of Cbl with p85 and leading to the coordinated activation of PI 3-kinase. However, since Lyn is also capable of phosphorylating Cbl in 293T cells and facilitating the physical association of p85 with Cbl, we cannot exclude the possibility that Lyn (and possibly Fgr) might also contribute to the tyrosine phosphorylation of Cbl and its association with p85 in LPS-stimulated Bac1.2F5 cells. The fact that a similar mechanism has been proposed for β1-integrin-mediated macrophage cell adhesion (
      • Meng F.
      • Lowell C.A.
      ) suggests that Src family kinases, Cbl, and PI 3-kinase may regulate the adherence of macrophages in response to various stimuli.
      Further investigation will be required to elucidate how activation of this Hck-Cbl-PI 3-kinase signal transduction pathway enhances the adherence of LPS-stimulated Bac1.2F5 cells. However, since the phosphorylation products of PI 3-kinase (e.g. PI 3,4,5-trisphosphate) can interact with a subset of pleckstrin homology domains (
      • Corvera S.
      • Czech M.P.
      ), it seems likely that a pleckstrin homology domain-containing protein might be involved in regulating the adherence of macrophages in response to LPS. Notably, Hmama et al.(
      • Hmama Z.
      • Knutson K.L.
      • Herrera-Velit P.
      • Nandan D.
      • Reiner N.E.
      ) have recently proposed a model in which LPS-induced monocyte adherence is mediated by the pleckstrin homology domain-containing protein cytohesin-1 (
      • Kolanus W.
      • Nagel W.
      • Schiller B.
      • Zeitlmann L.
      • Godar S.
      • Stockinger H.
      • Seed B.
      ,
      • Klarlund J.K.
      • Guilherme A.
      • Holik J.J.
      • Virbasius J.V.
      • Chawla A.
      • Czech M.P.
      ). Specifically, Hmama et al.have postulated that the activation of PI 3-kinase following LPS stimulation leads to the generation of PI 3,4,5-P3, which binds to, and modifies the properties of, cytohesin-1 (
      • Hmama Z.
      • Knutson K.L.
      • Herrera-Velit P.
      • Nandan D.
      • Reiner N.E.
      ). Engagement of the cytoplasmic tail of CD18 by cytohesin-1 would then lead to the conversion of low avidity LFA-1 (CD11a/CD18) molecules into high avidity molecules capable of increased binding to intercellular adhesion molecule 1 (
      • Hmama Z.
      • Knutson K.L.
      • Herrera-Velit P.
      • Nandan D.
      • Reiner N.E.
      ). This model for LPS-induced monocyte/macrophage cell adhesion can possibly now be extended further to incorporate the signal transduction events we have described in this report, namely that the activation of PI 3-kinase in response to LPS stimulation might be mediated by the phosphorylation of Cbl by Hck.
      We have been unable to maintain the phenotype of clonally derived Bac1-Hck499F cells. Upon extended passages, the cells (i) became morphology heterogeneous and (ii) exhibited levels of Hck activity and tyrosine phosphorylation of cellular proteins indistinguishable from that seen in Bac1.2F5 cells infected with the parental retrovirus.2 Thus, it has not been possible to ascertain if enforced expression of a constitutively activate form of Hck alone is sufficient to enhance the adherence of Bac1.2F5 cells. Additionally, it is unclear if tyrosine phosphorylation of Cbl (and its association with p85) alone accounts for the enhanced adherent properties of LPS-stimulated Bac1.2F5 cells. Intriguingly, we have detected partial translocation of Hck to the cytoskeleton of LPS-stimulated Bac1.2F5 cells. While the precise nature of this process remains to be established, it appears to be an F-actin-dependent process, since the actin-depolymerizing drug cytochalasin D perturbs Hck translocation.2 Additionally, far-Western blotting experiments revealed the presence of two proteins (p68 and p44) in the cytoskeletal fraction of Bac1.2F5 cells that bind Hck in an LPS-inducible fashion. However, the role these two proteins play, if any, in the translocation of Hck to the cytoskeleton is not known. Given that the cell's cytoskeleton is intimately involved in cell adhesion, it is tempting to speculate that in addition to the phosphorylation of Cbl, phosphorylation of cytoskeletal or cytoskeletally associated proteins by Hck may also contribute to enhancing the adherence of LPS-stimulated macrophages. Identification of additional substrates of Hck in LPS-stimulated Bac1.2F5 cells should provide further insight into the molecular mechanisms governing macrophage adhesion.

      Acknowledgments

      We thank Drs. Margaret Hibbs (Ludwig Institute for Cancer Research, Melbourne), Tom Gonda (Hanson Center for Cancer Research, Adelaide), Wallace Langdon (Department of Pathology, University of Western Australia), Manuela Baccarini (Institute for Microbiology and Genetics, Vienna), and Clifford Lowell (University of California, San Francisco) for gifts of various reagents. We also thank Professor Antony Burgess and Dr. Peter Lock for critical comments on the manuscript.

      REFERENCES

        • Auger M.J.
        • Ross J.A.
        Lewis C.E. McGee J.O.D. The Macrophage. IRL Press at Oxford University Press, Oxford1992: 1-74
        • Wright S.D.
        • Ramos R.A.
        • Tobias P.S.
        • Ulevitch R.J.
        • Mathison J.C.
        Science. 1990; 249: 1431-1433
        • Ulevitch R.J.
        • Tobias P.S.
        Curr. Opin. Immunol. 1994; 6: 125-130
        • Yang R.B.
        • Mark M.R.
        • Gray A.
        • Huang A.
        • Xie M.H.
        • Zhang M.
        • Goddard A.
        • Wood W.I.
        • Gurney A.L.
        • Godowski P.J.
        Nature. 1998; 395: 284-288
        • Kirschning C.J.
        • Wesche H.
        • Merrill-Ayres T.
        • Rothe M.
        J. Exp. Med. 1998; 188: 2091-2097
        • Heine H.
        • Kirschning C.J.
        • Lien E.
        • Monks B.G.
        • Rothe M.
        • Golenbock D.T.
        J. Immunol. 1999; 162: 6971-6975
        • Yang R.B.
        • Mark M.R.
        • Gurney A.L.
        • Godowski P.J.
        J. Immunol. 1999; 163: 639-643
        • Poltorak A.
        • He X.
        • Smirnova I.
        • Liu M.Y.
        • Huffel C.V.
        • Du X.
        • Birdwell D.
        • Alejos E.
        • Silva M.
        • Galanos C.
        • Freudenberg M.
        • Ricciardi-Castagnoli P.
        • Layton B.
        • Beutler B.
        Science. 1998; 282: 2085-2088
        • Chow J.C.
        • Young D.W.
        • Golenbock D.T.
        • Christ W.J.
        • Gusovsky F.
        J. Biol. Chem. 1999; 274: 10689-10692
        • Qureshi S.T.
        • Lariviere L.
        • Leveque G.
        • Clermont S.
        • Moore K.J.
        • Gros P.
        • Malo D.
        J. Exp. Med. 1999; 189: 615-625
        • Hoshino K.
        • Takeuchi O.
        • Kawai T.
        • Sanjo H.
        • Ogawa T.
        • Takeda Y.
        • Takeda K.
        • Akira S.
        J. Immunol. 1999; 162: 3749-3752
        • Rock F.L.
        • Hardiman G.
        • Timans J.C.
        • Kastelein R.A.
        • Bazan J.F.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593
        • Novotney M.
        • Chang Z.L.
        • Uchiyama H.
        • Suzuki T.
        Biochemistry. 1991; 30: 5597-5604
        • Shapira L.
        • Takashiba S.
        • Champagne C.
        • Amar S.
        • Van Dyke T.E.
        J. Immunol. 1994; 153: 1818-1824
        • Reimann T.
        • Buscher D.
        • Hipskind R.
        • Krautwald S.
        • Lohmann-Matthes M.L.
        • Baccarini M.
        J. Immunol. 1994; 153: 5740-5749
        • Weinstein S.L.
        • Sanghera J.S.
        • Lemke K.
        • DeFranco A.L.
        • Pelech S.L.
        J. Biol. Chem. 1992; 267: 14955-14962
        • Han J.
        • Lee J.D.
        • Bibbs L.
        • Ulevitch R.J.
        Science. 1994; 265: 808-811
        • Hambleton J.
        • Weinstein S.L.
        • Lem L.
        • DeFranco A.L.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2774-2778
        • Joseph C.K.
        • Wright S.D.
        • Bornmann W.G.
        • Randolph J.T.
        • Kumar E.R.
        • Bittman R.
        • Liu J.
        • Kolesnick R.N.
        J. Biol. Chem. 1994; 269: 17606-17610
        • Dong Z.
        • O'Brian C.A.
        • Fidler I.J.
        J. Leukocyte Biol. 1993; 53: 53-60
        • Geng Y.
        • Zhang B.
        • Lotz M.
        J. Immunol. 1993; 151: 6692-6700
        • Novogrodsky A.
        • Vanichkin A.
        • Patya M.
        • Gazit A.
        • Osherov N.
        • Levitzki A.
        Science. 1994; 264: 1319-1322
        • Stefanova I.
        • Corcoran M.L.
        • Horak E.M.
        • Wahl L.M.
        • Bolen J.B.
        • Horak I.D.
        J. Biol. Chem. 1993; 268: 20725-20728
        • English B.K.
        • Ihle J.N.
        • Myracle A.
        • Yi T.
        J. Exp. Med. 1993; 178: 1017-1022
        • Boulet I.
        • Ralph S.
        • Stanley E.
        • Lock P.
        • Dunn A.R.
        • Green S.P.
        • Phillips W.A.
        Oncogene. 1992; 7: 703-710
        • Lock P.
        • Stanley E.
        • Holtzman D.A.
        • Dunn A.R.
        Mol. Cell. Biol. 1990; 10: 4603-4611
        • Mizushima S.
        • Nagata S.
        Gene (Amst.). 1990; 18: 5322
        • Rayner J.R.
        • Gonda T.J.
        Mol. Cell. Biol. 1994; 14: 880-887
        • Boussif O.
        • Lezoualc'h F.
        • Zanta M.A.
        • Mergny M.D.
        • Scherman D.
        • Demeneix B.
        • Behr J.P.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301
        • Smith D.B.
        • Johnson K.S.
        Gene (Amst.). 1988; 67: 31-40
        • Morgan C.
        • Pollard J.W.
        • Stanley E.R.
        J. Cell. Physiol. 1987; 130: 420-427
        • Buscher D.
        • Sbarba P.D.
        • Hipskind R.A.
        • Rapp U.R.
        • Stanley E.R.
        • Baccarini M.
        Oncogene. 1993; 8: 3323-3332
        • Odai H.
        • Sasaki K.
        • Iwamatsu A.
        • Hanazono Y.
        • Tanaka T.
        • Mitani K.
        • Yazaki Y.
        • Hirai H.
        J. Biol. Chem. 1995; 270: 10800-10805
        • Wang Y.
        • Yeung Y.G.
        • Langdon W.Y.
        • Stanley E.R.
        J. Biol. Chem. 1996; 271: 17-20
        • Anderson S.M.
        • Burton E.A.
        • Koch B.L.
        J. Biol. Chem. 1997; 272: 739-745
        • Barber D.L.
        • Mason J.M.
        • Fukazawa T.
        • Reedquist K.A.
        • Druker B.J.
        • Band H
        • D'Andrea A.D.
        Blood. 1997; 89: 3166-3174
        • Ueno H.
        • Sasaki K.
        • Honda H.
        • Nakamoto T.
        • Yamagata T.
        • Miyagawa K.
        • Mitani K.
        • Yazaki Y.
        • Hirai H.
        Blood. 1998; 91: 46-53
        • Donovan J.A.
        • Wange R.L.
        • Langdon W.Y.
        • Samelson L.E.
        J. Biol. Chem. 1994; 269: 22921-22924
        • Meisner H.
        • Conway B.R.
        • Hartley D.
        • Czech M.P.
        Mol. Cell. Biol. 1995; 15: 3571-3578
        • Fukazawa T.
        • Reedquist K.A.
        • Trub T.
        • Soltoff S.
        • Panchamoorthy G.
        • Druker B.
        • Cantley L.
        • Shoelson S.E.
        • Band H.
        J. Biol. Chem. 1995; 270: 19141-19150
        • Buday L.
        • Khwaja A.
        • Sipeki S.
        • Farago A.
        • Downward J.
        J. Biol. Chem. 1996; 271: 6159-6163
        • Panchamoorthy G.
        • Fukazawa T.
        • Miyake S.
        • Soltoff S.
        • Reedquist K.
        • Druker B
        • Schoelson S.
        • Cantley L.
        • Band H.
        J. Biol. Chem. 1996; 271: 3187-3194
        • Ojaniemi M.
        • Martin S.S.
        • Dolfi F.
        • Olefsky J.M.
        • Vuori K.
        J. Biol. Chem. 1997; 272: 3780-3787
        • Andoniou C.E.
        • Thien C.B.
        • Langdon W.Y.
        EMBO J. 1994; 13: 4515-454523
        • Ribon V.
        • Hubbell S.
        • Herrera R.
        • Saltiel A.R.
        Mol. Cell. Biol. 1996; 16: 45-52
        • Hunter S.
        • Burton E.A.
        • Wu S.C.
        • Anderson S.M.
        J. Biol. Chem. 1999; 274: 2097-2106
        • Meng F.
        • Lowell C.A.
        EMBO J. 1998; 17: 4391-4403
        • Howlett C.J.
        • Bisson S.A.
        • Resek M.E.
        • Tigley A.W.
        • Robbins S.M.
        Biochem. Biophys. Res. Commun. 1999; 257: 129-138
        • Sicheri F.
        • Moarefi I.
        • Kuriyan J.
        Nature. 1997; 385: 602-609
        • Horita D.A.
        • Baldisseri D.M.
        • Zhang W.
        • Altieri A.S.
        • Smithgall T.E.
        • Gmeiner W.H.
        • Byrd R.A.
        J. Mol. Biol. 1998; 278: 253-265
        • Marcilla A.
        • Rivero-Lezcano O.M.
        • Agarwal A.
        • Robbins K.C.
        J. Biol. Chem. 1995; 270: 9115-9120
        • 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.C.
        Cell. 1993; 72: 767-778
        • Meng F.
        • Lowell C.A.
        J. Exp. Med. 1997; 185: 1661-1670
        • Chiaradonna F.
        • Fontana L.
        • Iavarone C.
        • Carriero M.V.
        • Scholz G.
        • Barone M.V.
        • Stoppelli M.P.
        EMBO J. 1999; 18: 3013-3023
        • Corvera S.
        • Czech M.P.
        Trends Cell Biol. 1998; 8: 442-446
        • Hmama Z.
        • Knutson K.L.
        • Herrera-Velit P.
        • Nandan D.
        • Reiner N.E.
        J. Biol. Chem. 1999; 274: 1050-1057
        • Kolanus W.
        • Nagel W.
        • Schiller B.
        • Zeitlmann L.
        • Godar S.
        • Stockinger H.
        • Seed B.
        Cell. 1996; 86: 233-242
        • Klarlund J.K.
        • Guilherme A.
        • Holik J.J.
        • Virbasius J.V.
        • Chawla A.
        • Czech M.P.
        Science. 1997; 275: 1927-1930