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Phosphatidylinositol 3-Kinase and Ca2+ Influx Dependence for Ligand-stimulated Internalization of the c-Kit Receptor*

  • Jennifer L. Gommerman
    Affiliations
    Wellesley Hospital Research Institute and Department of Immunology, University of Toronto, Toronto, Ontario, Canada M4Y 1J3
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  • Robert Rottapel
    Affiliations
    Wellesley Hospital Research Institute and Department of Immunology, University of Toronto, Toronto, Ontario, Canada M4Y 1J3
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  • Stuart A. Berger
    Correspondence
    To whom correspondence should be addressed: Wellesley Hospital Research Institute, 160 Wellesley St. E., Toronto, Ontario, Canada M4Y 1J3. Tel.: 416-926-5148; Fax: 416-926-5109
    Affiliations
    Wellesley Hospital Research Institute and Department of Immunology, University of Toronto, Toronto, Ontario, Canada M4Y 1J3
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  • Author Footnotes
    * This work was supported by a grant from the National Cancer Institute of Canada (to S. A. B.).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:November 28, 1997DOI:https://doi.org/10.1074/jbc.272.48.30519
      We have evaluated the role of phosphatidylinositol 3-kinase (PI3-kinase) and Ca2+influx in ligand-stimulated internalization of the c-Kit receptor. The wild type (wt) c-Kit receptor and YF719, a mutant receptor in which the SH2-mediated binding site for the p85 subunit of PI3-kinase is disrupted, were expressed in DA-1 cells. YF719 internalized with similar kinetics as wt c-Kit although the receptor remained localized close to the plasma membrane. However, in the absence of extracellular Ca2+, or in the presence of the competitive Ca2+ influx blocker Ni2+, the YF719 mutant failed to internalize. Failure to internalize in the absence of Ca2+ was also observed for the wt c-Kit receptor in cells that were pretreated with the PI3-kinase inhibitor, wortmannin. Following stimulation with ligand, clathrin heavy chains were found to co-immunoprecipitate with c-Kit. However, under conditions in which PI3-kinase activity is inhibited and Ca2+ influx is blocked, clathrin failed to co-immunoprecipitate with c-Kit. Our results demonstrate that both Ca2+ influx and PI3-kinase activity influence c-Kit endocytosis, and inhibition of these two signals disrupts the earliest stages of ligand-mediated internalization.
      c-Kit is a receptor tyrosine kinase and a member of the subfamily that includes the PDGF,
      The abbreviations used are: PDGF, platelet-derived growth factor; SLF, steel factor; PI3-kinase, phosphatidylinositol 3-kinase; BMMC, bone marrow-derived mast cells; wt, wild type; PBS, phosphate-buffered saline; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; TxR, Texas Red.
      1The abbreviations used are: PDGF, platelet-derived growth factor; SLF, steel factor; PI3-kinase, phosphatidylinositol 3-kinase; BMMC, bone marrow-derived mast cells; wt, wild type; PBS, phosphate-buffered saline; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; TxR, Texas Red.
      CSF-1, and flt-3/flk-2 receptors. Together with its ligand steel factor (SLF), c-Kit is a key controlling receptor for a number of cell types including hematopoietic stem cells, mast cells, melanocytes, and germ cells. c-Kit is the gene product of the W locus in mice (
      • Huang E.
      • Nocka K.
      • Beier D.R.
      • Chu T.Y.
      • Buck J.
      • Lahm H.W.
      • Wellner D.
      • Leder P.
      • Besmer P.
      ), and its ligand SLF is the product of the sl locus (
      • Williams D.E.
      • Eisenman J.
      • Baird A.
      • Rauch C.
      • Van N.K.
      • March C.J.
      • Park L.S.
      • Martin U.
      • Mochizuki D.Y.
      • Boswell H.S.
      • Burgess G.S.
      • Cosman D.
      • Lyman S.D.
      ,
      • Zsebo K.M.
      • Williams D.A.
      • Geissler E.N.
      • Broudy V.C.
      • Martin F.H.
      • Atkins H.L.
      • Hsu R.Y.
      • Birkett N.C.
      • Okino K.H.
      • Murdock D.C.
      • Jacobsen F.W.
      • Langley K.E.
      • Smith K.A.
      • Takeishi T.
      • Cattanach B.M.
      • Galli S.J.
      • Suggs S.V.
      ). Mutations in either locus severely affect the growth and survival of stem cells of these tissues.
      As with other receptor tyrosine kinases, stimulation of the c-Kit receptor with SLF results in the recruitment and tyrosine phosphorylation of SH2-containing second messenger-generating enzymes (
      • Ullrich A.
      • Schlessinger J.
      ) such as phospholipase C-γ and PI3-kinase (
      • Rottapel R.
      • Reedijk M.
      • Williams D.E.
      • Lyman S.D.
      • Anderson D.M.
      • Pawson T.
      • Bernstein A.
      ,
      • Lev S.
      • Givol D.
      • Yarden Y.
      ). The phosphorylated lipid products of these enzymes stimulate a variety of intracellular processes including Ca2+ mobilization and actin reorganization (
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson W.L.
      • Stephens L.
      ,
      • Fukamachi H.
      • Kawakami Y.
      • Takei M.
      • Ishizaka T.
      • Ishizaka K.
      • Kawakami T.
      ,
      • Catt K.J.
      • Hunyady L.
      • Balla T.
      ). Coincident with second messenger generation, the process of receptor internalization is also initiated. Within minutes following ligand binding, receptors cluster in dimers or oligomers and internalize by endocytosis, likely through clathrin-coated pits (
      • Sorkin A.
      • Carpenter G.
      ,
      • Schmid S.L.
      • Damke H.
      ,
      • Robinson M.S.
      ). Eventually, clathrin coats are removed, and the remaining vesicles fuse with endosomes, late endosomes, and ultimately lysosomes, resulting in receptor degradation.
      Numerous deletion and mutagenesis studies have been carried out to map regions of receptor tyrosine kinases required for ligand-stimulated internalization (
      • Lin C.R.
      • Chen W.S.
      • Lazar C.S.
      • Carpenter C.D.
      • Gill G.N.
      • Evans R.M.
      • Rosenfeld M.G.
      ,
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ,
      • Mori S.
      • Ronnstrand L.
      • Claesson-Welsh L.
      • Heldin C.H.
      ,
      • Severinsson L.
      • Ek B.
      • Mellstrom K.
      • Claesson W.L.
      • Heldin C.H.
      ,
      • Glenney J.J.
      • Chen W.S.
      • Lazar C.S.
      • Walton G.M.
      • Zokas L.M.
      • Rosenfeld M.G.
      • Gill G.N.
      ,
      • Chen W.S.
      • Lazar C.S.
      • Lund K.A.
      • Welsh J.B.
      • Chang C.P.
      • Walton G.M.
      • Der C.J.
      • Wiley H.S.
      • Gill G.N.
      • Rosenfeld M.G.
      ,
      • Chang C.P.
      • Lazar C.S.
      • Walsh B.J.
      • Komuro M.
      • Collawn J.F.
      • Kuhn L.A.
      • Tainer J.A.
      • Trowbridge I.S.
      • Farquhar M.G.
      • Rosenfeld M.G.
      • Wiley H.S.
      • Gill G.N.
      ,
      • Dargemont C.
      • Le B.A.
      • Rothenberger S.
      • Iacopetta B.
      • Kuhn L.C.
      ,
      • Myles G.M.
      • Brandt C.S.
      • Carlberg K.
      • Rohrschneider L.R.
      ,
      • Yamada K.
      • Carpentier J.L.
      • Cheatham B.
      • Goncalves E.
      • Shoelson S.E.
      • Kahn C.R.
      ). Tyrosine kinase activity (
      • Lamaze C.
      • Schmid S.L.
      ,
      • Downing J.R.
      • Roussel M.F.
      • Sherr C.J.
      ), autophosphorylation sites (
      • Glenney J.J.
      • Chen W.S.
      • Lazar C.S.
      • Walton G.M.
      • Zokas L.M.
      • Rosenfeld M.G.
      • Gill G.N.
      ,
      • Chen W.S.
      • Lazar C.S.
      • Lund K.A.
      • Welsh J.B.
      • Chang C.P.
      • Walton G.M.
      • Der C.J.
      • Wiley H.S.
      • Gill G.N.
      • Rosenfeld M.G.
      ), and interactions with second messenger-generating enzymes have been implicated. In particular, recruitment and activation of PI3-kinase have been associated with ligand-stimulated internalization of the PDGF receptor (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ). Receptors carrying mutations within the cytoplasmic domain that disrupt the PI3-kinase-binding site are impaired in the later stages of endocytosis (
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ). However, conflicting results with a deletion mutant encompassing this binding site demonstrate no impairment in any stage of endocytosis (
      • Severinsson L.
      • Ek B.
      • Mellstrom K.
      • Claesson W.L.
      • Heldin C.H.
      ).
      Ligand-stimulated endocytosis of c-Kit was investigated by Yee et al. (
      • Yee N.S.
      • Hsiau C.W.
      • Serve H.
      • Vosseller K.
      • Besmer P.
      ). They reported that a c-Kit mutant with no kinase activity was impaired for ligand-stimulated internalization. Individual mutations converting tyrosines 719 and 821 to phenylalanines, however, failed to affect internalization, although Phe-821 was impaired for mitogenesis (
      • Serve H.
      • Yee N.S.
      • Stella G.
      • Sepp L.L.
      • Tan J.C.
      • Besmer P.
      ). Tyr-719 is located in the kinase insert region of the c-Kit catalytic domain. In its phosphorylated form, it forms part of a consensus binding site for the p85 subunit of PI3-kinase and has been shown to mediate this interaction in vitro and in vivo (
      • Lev S.
      • Givol D.
      • Yarden Y.
      ).
      We have also examined the role of PI3-kinase activation on ligand-stimulated internalization of c-Kit. We found that in the absence of PI3-kinase activation, the c-Kit receptor internalizes but remains localized near the inner aspect of the plasma membrane. However, when both PI3-kinase and Ca2+ influx are inhibited, clathrin fails to co-immunoprecipitate with c-Kit, and receptor internalization is completely prevented. These results show that concurrent inhibition of PI3-kinase activity and Ca2+influx disrupts the earliest stages of c-Kit internalization.

      DISCUSSION

      Ligand-stimulated receptor internalization is a multi-step process involving assembly of an endocytic machinery composed of clathrin heavy and light chains, adaptors, dynamin, and other cytosolic factors. In addition to these elements, recruitment and activation of enzymes such as PI3-kinase have also been implicated in the endocytic process. We found that ligand-stimulated internalization of the c-Kit receptor is blocked when both PI3-kinase activation and Ca2+ influx are inhibited. Although inhibition of either of these signals alone did not prevent the early stages of internalization, loss of PI3-kinase activity resulted in internalized receptors that appeared to accumulate in vesicles close to the membrane. This observation is consistent with the results of Joly et al. (
      • Joly M.
      • Kazlauskas A.
      • Corvera S.
      ) who demonstrated that a mutant PDGF receptor that cannot associate with PI3-kinase is internalized but not degraded.
      PI3-kinase is a second messenger-generating enzyme that has been linked to mitogenesis, receptor trafficking, and maintenance of cell viability in other receptor systems (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ,
      • Serve H.
      • Yee N.S.
      • Stella G.
      • Sepp L.L.
      • Tan J.C.
      • Besmer P.
      ,
      • Yao R.
      • Cooper G.M.
      ). Indirect evidence suggests a role for PI3-kinase in vesicular sorting or movement of proteins in the cell. Vps34, a yeast protein with homology to the p110 catalytic subunit of PI3-kinase, is involved in the transport of soluble hydrolases from the trans Golgi network to yeast vacuoles (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ). Kapeller and Cantley (
      • Kapeller R.
      • Cantley L.C.
      ) have shown that activated PDGF receptor and PI3-kinase remained complexed in endosomes and associate with microtubules in 3T3-L1 cells. Inhibition of PI3-kinase has also been reported to inhibit transferrin receptor endocytosis, nonspecific fluid phase uptake, and early endosome fusion, possibly via the small GTPase Rab5 (
      • Li G.
      • D'Souza S.C.
      • Barbieri M.A.
      • Roberts R.L.
      • Klippel A.
      • Williams L.T.
      • Stahl P.D.
      ). In addition, PI3-kinase activation is required for the reorganization of actin filaments and the induction of membrane ruffling by PDGF (
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson W.L.
      • Stephens L.
      ). Besmer and colleagues (
      • Yee N.S.
      • Hsiau C.W.
      • Serve H.
      • Vosseller K.
      • Besmer P.
      ) found little to no effect of a YF719 c-Kit mutation on ligand-stimulated internalization. However, the internalization experiments reported by these researchers were performed under conditions permitting Ca2+ influx, which our results show are critical for internalization in the absence of PI3-kinase.
      Although clathrin-independent endocytosis has been reported (
      • Sorkin A.
      • Carpenter G.
      ), our observation that clathrin co-immunoprecipitates with c-Kit following stimulation with ligand indicates that internalization of this receptor is clathrin-associated. Importantly, inhibition of either PI3-kinase or Ca2+ influx alone did not prevent this association, suggesting that under either of these conditions, c-Kit internalization remains clathrin-associated. We did, however, fail to observe clathrin co-immunoprecipitation with c-Kit when both PI3-kinase and Ca2+ influx were inhibited. This result indicates that the internalization block under these conditions may be occurring at the earliest step of clathrin cage formation.
      c-Kit activates phospholipase C-γ (
      • Rottapel R.
      • Reedijk M.
      • Williams D.E.
      • Lyman S.D.
      • Anderson D.M.
      • Pawson T.
      • Bernstein A.
      ,
      • Fukamachi H.
      • Kawakami Y.
      • Takei M.
      • Ishizaka T.
      • Ishizaka K.
      • Kawakami T.
      ) resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate (
      • Catt K.J.
      • Hunyady L.
      • Balla T.
      ). Inositol 1,4,5-trisphosphate causes the mobilization of intracellular Ca2+ from stores in the endoplasmic reticulum, resulting in the stimulation of Ca2+influx from the extracellular milieu through the Ca2+release activated channel (
      • Hoth M.
      • Penner R.
      ). This Ca2+ influx step seems to be critical for c-Kit internalization, since intracellular mobilization should still occur in the absence of extracellular Ca2+ or in the presence of Ni2+.
      Our results do not identify the Ca2+-dependent step in the early stages of receptor endocytosis. However, a number of Ca2+-dependent elements implicated in endocytosis have been identified. Clathrin light chains are Ca2+-binding proteins, and high levels of Ca2+are thought to stabilize the clathrin cage (
      • Mooibroek M.J.
      • Michiel D.F.
      • Wang J.H.
      ,
      • Nathke I.
      • Hill B.L.
      • Parham P.
      • Brodsky F.M.
      ). The adaptor protein annexin VI, a Ca2+-dependent phospholipid-binding protein, is required for budding of clathrin-coated pits in a cell-free system (
      • Lin H.T.
      • Sudhof T.C.
      • Anderson R.G.W.
      ). However, annexin VI was not found to have a role in coated pit formation and constriction nor did it enhance transferrin receptor endocytosis in A431 cells (
      • Smythe E.
      • Smith P.D.
      • Jacob S.M.
      • Theobald J.
      • Moss S.E.
      ). Another Ca2+-dependent protein is calmodulin, a cytoplasmic mediator of many calcium-regulated processes (
      • Takuwa N.
      • Zhou W.
      • Takuwa Y.
      ). Calmodulin has been demonstrated to bind to clathrin light chains in a Ca2+-dependent manner (
      • Pley U.M.
      • Hill B.L.
      • Alibert C.
      • Brodsky F.M.
      • Parham P.
      ), has been shown to regulate endosome fusion in vitro (
      • Colombo M.I.
      • Beron W.
      • Stahl P.D.
      ), and is implicated in other membrane trafficking events (
      • Apodaca G.
      • Enrich C.
      • Mostov K.E.
      ). Future experiments will be required to more fully determine the role of these elements in the Ca2+-sensitive phase of c-Kit internalization.

      Acknowledgments

      We thank N. Casteran for transfecting the expression vectors into the gp + e cells, R. Chow for assistance in preparing recombinant SLF, and J. LaRose for technical advice with respect to immunoprecipitations.

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