FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1979541028> ?p ?o ?g. }

• W1979541028 endingPage "5403" @default.
• W1979541028 startingPage "5392" @default.
• W1979541028 abstract "Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle. Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle. IntroductionDisabled-2 (Dab2) is a multidomain mitotic phosphoprotein that functions as a signal modulator and tumor suppressor (1Wang Z. Tseng C.P. Pong R.C. Chen H. McConnell J.D. Navone N. Hsieh J.T. J. Biol. Chem. 2002; 277: 12622-12631Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 2Cho S.Y. Jeon J.W. Lee S.H. Park S.S. Biochem. J. 2000; 352: 645-650Crossref PubMed Scopus (23) Google Scholar, 3Huang C.H. Cheng J.C. Chen J.C. Tseng C.P. Cell. Signal. 2007; 19: 1339-1347Crossref PubMed Scopus (22) Google Scholar, 4Xu X.X. Yi T. Tang B. Lambeth J.D. Oncogene. 1998; 16: 1561-1569Crossref PubMed Scopus (106) Google Scholar, 5Zhou J. Hsieh J.T. J. Biol. Chem. 2001; 276: 27793-27798Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Zhou J. Scholes J. Hsieh J.T. J. Biol. Chem. 2003; 278: 6936-6941Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Jiang Y. Prunier C. Howe P.H. Oncogene. 2008; 27: 1865-1875Crossref PubMed Scopus (44) Google Scholar, 8Hocevar B.A. Mou F. Rennolds J.L. Morris S.M. Cooper J.A. Howe P.H. EMBO J. 2003; 22: 3084-3094Crossref PubMed Scopus (113) Google Scholar, 9Hocevar B.A. Prunier C. Howe P.H. J. Biol. Chem. 2005; 280: 25920-25927Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 10Hocevar B.A. Smine A. Xu X.X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar, 11Prunier C. Howe P.H. J. Biol. Chem. 2005; 280: 17540-17548Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and as a cargo-specific adaptor of clathrin-mediated endocytosis (12Yang D.H. Smith E.R. Roland I.H. Sheng Z. He J. Martin W.D. Hamilton T.C. Lambeth J.D. Xu X.X. Dev. Biol. 2002; 251: 27-44Crossref PubMed Scopus (137) Google Scholar, 13Keyel P.A. Mishra S.K. Roth R. Heuser J.E. Watkins S.C. Traub L.M. Mol. Biol. Cell. 2006; 17: 4300-4317Crossref PubMed Scopus (109) Google Scholar, 14Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (249) Google Scholar, 15Maurer M.E. Cooper J.A. J. Cell Sci. 2006; 119: 4235-4246Crossref PubMed Scopus (139) Google Scholar, 16Maurer M.E. Cooper J.A. J. Cell Sci. 2005; 118: 5345-5355Crossref PubMed Scopus (80) Google Scholar, 17Nagai J. Christensen E.I. Morris S.M. Willnow T.E. Cooper J.A. Nielsen R. Am. J. Physiol. Renal Physiol. 2005; 289: F569-F576Crossref PubMed Scopus (81) Google Scholar, 18Morris S.M. Arden S.D. Roberts R.C. Kendrick-Jones J. Cooper J.A. Luzio J.P. Buss F. Traffic. 2002; 3: 331-341Crossref PubMed Scopus (198) Google Scholar, 19Morris S.M. Tallquist M.D. Rock C.O. Cooper J.A. EMBO J. 2002; 21: 1555-1564Crossref PubMed Scopus (173) Google Scholar, 20Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar, 21Cuitino L. Matute R. Retamal C. Bu G. Inestrosa N.C. Marzolo M.P. Traffic. 2005; 6: 820-838Crossref PubMed Scopus (58) Google Scholar). Different Dab2 isoforms localize to distinct intracellular compartments. Thus, at steady state, the long isoform (denominated p96 or p82 in different species) localizes to the plasma membrane (14Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (249) Google Scholar, 18Morris S.M. Arden S.D. Roberts R.C. Kendrick-Jones J. Cooper J.A. Luzio J.P. Buss F. Traffic. 2002; 3: 331-341Crossref PubMed Scopus (198) Google Scholar, 20Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar), whereas the short isoform (denominated p67 or p59), which is devoid of a central exon (22Xu X.X. Yang W. Jackowski S. Rock C.O. J. Biol. Chem. 1995; 270: 14184-14191Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), presents a diffuse distribution pattern with a considerable localization to the cell nucleus (2Cho S.Y. Jeon J.W. Lee S.H. Park S.S. Biochem. J. 2000; 352: 645-650Crossref PubMed Scopus (23) Google Scholar). In accord with its multiple functions, a number of interactors have been identified for Dab2. These include clathrin (14Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (249) Google Scholar), the adaptor complex 2 (AP2 (20Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar)), NPXY-containing cargo (13Keyel P.A. Mishra S.K. Roth R. Heuser J.E. Watkins S.C. Traub L.M. Mol. Biol. Cell. 2006; 17: 4300-4317Crossref PubMed Scopus (109) Google Scholar, 14Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (249) Google Scholar, 15Maurer M.E. Cooper J.A. J. Cell Sci. 2006; 119: 4235-4246Crossref PubMed Scopus (139) Google Scholar, 16Maurer M.E. Cooper J.A. J. Cell Sci. 2005; 118: 5345-5355Crossref PubMed Scopus (80) Google Scholar, 20Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar), and myosin VI (18Morris S.M. Arden S.D. Roberts R.C. Kendrick-Jones J. Cooper J.A. Luzio J.P. Buss F. Traffic. 2002; 3: 331-341Crossref PubMed Scopus (198) Google Scholar, 23Hasson T. J. Cell Sci. 2003; 116: 3453-3461Crossref PubMed Scopus (141) Google Scholar, 24Dance A.L. Miller M. Seragaki S. Aryal P. White B. Aschenbrenner L. Hasson T. Traffic. 2004; 5: 798-813Crossref PubMed Scopus (84) Google Scholar, 25Spudich G. Chibalina M.V. Au J.S. Arden S.D. Buss F. Kendrick-Jones J. Nat. Cell Biol. 2007; 9: 176-183Crossref PubMed Scopus (161) Google Scholar), all related to its endocytic functions, and Dab2-interacting protein (Dab2IP (1Wang Z. Tseng C.P. Pong R.C. Chen H. McConnell J.D. Navone N. Hsieh J.T. J. Biol. Chem. 2002; 277: 12622-12631Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar)), Src (6Zhou J. Scholes J. Hsieh J.T. J. Biol. Chem. 2003; 278: 6936-6941Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), Grb (4Xu X.X. Yi T. Tang B. Lambeth J.D. Oncogene. 1998; 16: 1561-1569Crossref PubMed Scopus (106) Google Scholar), CIN85 (26Kowanetz K. Terzic J. Dikic I. FEBS Lett. 2003; 554: 81-87Crossref PubMed Scopus (33) Google Scholar), axin (7Jiang Y. Prunier C. Howe P.H. Oncogene. 2008; 27: 1865-1875Crossref PubMed Scopus (44) Google Scholar), Smads, and the transforming growth factor β (TGF-β) receptors (9Hocevar B.A. Prunier C. Howe P.H. J. Biol. Chem. 2005; 280: 25920-25927Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 10Hocevar B.A. Smine A. Xu X.X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar), which enable the signal-modulating potential of Dab2. Dab2 also binds directly to integrins and may thus coordinate changes to cell adhesion, cell motility, membrane trafficking, and signaling (11Prunier C. Howe P.H. J. Biol. Chem. 2005; 280: 17540-17548Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 27Chao W.T. Kunz J. FEBS Lett. 2009; 583: 1337-1343Crossref PubMed Scopus (146) Google Scholar, 28Huang C.L. Cheng J.C. Stern A. Hsieh J.T. Liao C.H. Tseng C.P. J. Cell Sci. 2006; 119: 4420-4430Crossref PubMed Scopus (30) Google Scholar, 29Calderwood D.A. Fujioka Y. de Pereda J.M. García-Alvarez B. Nakamoto T. Margolis B. McGlade C.J. Liddington R.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2272-2277Crossref PubMed Scopus (333) Google Scholar, 30Teckchandani A. Toida N. Goodchild J. Henderson C. Watts J. Wollscheid B. Cooper J.A. J. Cell Biol. 2009; 186: 99-111Crossref PubMed Scopus (91) Google Scholar). Taken together, the current picture suggests that the function of Dab2 is regulated by intracellular localization, by association with specific interacting factors, and possibly by post-translational modification. However, the interdependence of these different modes of regulation still remains to be determined and is at the center of this study.The mitotic shutdown of endocytosis, a mechanism proposed more than 25 years ago (31Warren G. Davoust J. Cockcroft A. EMBO J. 1984; 3: 2217-2225Crossref PubMed Scopus (81) Google Scholar, 32Pypaert M. Lucocq J.M. Warren G. Eur. J. Cell Biol. 1987; 45: 23-29PubMed Google Scholar, 33Pypaert M. Mundy D. Souter E. Labbé J.C. Warren G. J. Cell Biol. 1991; 114: 1159-1166Crossref PubMed Scopus (49) Google Scholar), is a contentious subject (32Pypaert M. Lucocq J.M. Warren G. Eur. J. Cell Biol. 1987; 45: 23-29PubMed Google Scholar, 33Pypaert M. Mundy D. Souter E. Labbé J.C. Warren G. J. Cell Biol. 1991; 114: 1159-1166Crossref PubMed Scopus (49) Google Scholar, 34Schweitzer J.K. Burke E.E. Goodson H.V. D'Souza-Schorey C. J. Biol. Chem. 2005; 280: 41628-41635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 35Boucrot E. Kirchhausen T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7939-7944Crossref PubMed Scopus (249) Google Scholar, 36Raucher D. Sheetz M.P. J. Cell Biol. 1999; 144: 497-506Crossref PubMed Scopus (172) Google Scholar). However, a consensus exists concerning the re-localization of clathrin to the mitotic spindle (37Borlido J. Veltri G. Jackson A.P. Mills I.G. PLoS ONE. 2008; 3: e3115Crossref PubMed Scopus (8) Google Scholar, 38Yamauchi T. Ishidao T. Nomura T. Shinagawa T. Tanaka Y. Yonemura S. Ishii S. EMBO J. 2008; 27: 1852-1862Crossref PubMed Scopus (45) Google Scholar, 39Royle S.J. Bright N.A. Lagnado L. Nature. 2005; 434: 1152-1157Crossref PubMed Scopus (197) Google Scholar, 40Okamoto C.T. McKinney J. Jeng Y.Y. Am. J. Physiol. Cell Physiol. 2000; 279: C369-C374Crossref PubMed Google Scholar), a shutdown of recycling at prophase, and its renewal and functional importance in cytokinesis (34Schweitzer J.K. Burke E.E. Goodson H.V. D'Souza-Schorey C. J. Biol. Chem. 2005; 280: 41628-41635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 41Albertson R. Riggs B. Sullivan W. Trends Cell Biol. 2005; 15: 92-101Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The notion of a modulation of the functions of endocytic proteins in mitosis is supported by the mitosis-related post-translational modifications of these proteins. Thus, Dab2, epsin, and Eps15 were proposed to undergo a transient cdc2-mediated phosphorylation in mitosis (42Chen H. Slepnev V.I. Di Fiore P.P. De Camilli P. J. Biol. Chem. 1999; 274: 3257-3260Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 43He J. Xu J. Xu X.X. Hall R.A. Oncogene. 2003; 22: 4524-4530Crossref PubMed Scopus (30) Google Scholar). Furthermore, an alteration in the intracellular localization of Dab2 in mitosis has been suggested (14Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (249) Google Scholar). However, a thorough description of the cell cycle-dependent alterations to the localization and functional interactions of Dab2 and an assessment of the endocytosis of cargo endowed with different internalization signals in mitosis are currently lacking and will be addressed here.The spindle assembly checkpoint (SAC) 2The abbreviations used are: SACspindle assembly checkpointCCFcorrelation coefficientLDLRlow density lipoprotein receptorTβRItype I TGF-β receptorVtMvariance to mean ratio2ME22-methoxyestradiolhhumanrrat. is a critical step in the transition from the early to late stages of mitosis (44Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1702) Google Scholar). Low concentrations of nocodazole, vinblastine, or 2-methoxyestradiol (2ME2) induce an arrest of the cell cycle at the SAC without causing massive microtubule depolymerization (45Jordan M.A. Thrower D. Wilson L. J. Cell Sci. 1992; 102: 401-416Crossref PubMed Google Scholar, 46Attalla H. Mäkelä T.P. Adlercreutz H. Andersson L.C. Biochem. Biophys. Res. Commun. 1996; 228: 467-473Crossref PubMed Scopus (112) Google Scholar, 47Kamath K. Okouneva T. Larson G. Panda D. Wilson L. Jordan M.A. Mol. Cancer Ther. 2006; 5: 2225-2233Crossref PubMed Scopus (74) Google Scholar). Cytosol extracts, of cells arrested in this manner, accumulate mitotically phosphorylated endocytic proteins and hamper the invagination of clathrin-coated pits in permeabilized cells (32Pypaert M. Lucocq J.M. Warren G. Eur. J. Cell Biol. 1987; 45: 23-29PubMed Google Scholar, 33Pypaert M. Mundy D. Souter E. Labbé J.C. Warren G. J. Cell Biol. 1991; 114: 1159-1166Crossref PubMed Scopus (49) Google Scholar, 42Chen H. Slepnev V.I. Di Fiore P.P. De Camilli P. J. Biol. Chem. 1999; 274: 3257-3260Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 43He J. Xu J. Xu X.X. Hall R.A. Oncogene. 2003; 22: 4524-4530Crossref PubMed Scopus (30) Google Scholar).Here, we employ ES-2 ovary cancer cells and 2ME2 to show that the intracellular localization of the p96/p82 isoform of Dab2, its phosphorylation state, and its association with clathrin are cell cycle-dependent. Moreover, in G2/M-arrested cells, the internalization of endocytic cargo endowed with an NPXY signal is inhibited.DISCUSSIONThe notion that the intracellular localization, repertoire of interactions, and function of Dab2 are altered in mitosis is supported by the following lines of evidence: (i) in interphase cells, the intracellular distribution of Dab2 yields a punctate staining characterized by a high variance to mean ratio (VtM); this staining pattern is lost throughout the mitosis of cycling cells and in cells arrested at the SAC with 2ME2, where a diffuse staining pattern is characterized by a low VtM. Importantly, the VtM obtained under these latter conditions is similar to the one observed in the interior of the cell, further supporting the notion of the displacement of Dab2 to the cytosol in mitosis (Fig. 1 and supplemental Fig. 1). Moreover, the distribution pattern of Dab2 in cells in cytokinesis yields an intermediate VtM value (Fig. 1 and supplemental Fig. 1), stressing the cyclical nature of the displacement of Dab2 in the cell cycle. The notion of the displacement of Dab2 to the cytosol is further supported by the 2ME2-induced alterations observed by cell fractionation (supplemental Fig. 2). (ii) At the plasma membrane of interphase cells, Dab2 and clathrin show a high degree of co-localization, reflected by the high value of the maximal Pearson's CCF of their fluorescence signals. In the course of mitosis, this CCF is significantly reduced (Fig. 2G). Here too, 2ME2-mediated cell cycle arrest emulated the effects observed in mitotic/cycling cells (supplemental Fig. 3B), reinforcing the notion of its appropriateness as a means of enrichment of the mitotic cell population. Importantly, in mitosis, clathrin and Dab2 accumulated in different intracellular compartments. Although clathrin showed a prominent recruitment to the mitotic spindle, Dab2 remained diffuse in the cytoplasm of the cell (FIGURE 2, FIGURE 3B). Here too, the CCF value obtained in cytokinesis is significantly higher than the minimal value, observed in cells in metaphase (Fig. 2G), reinforcing the notion of the cyclical regulation of Dab2/clathrin interactions. (iii) Dab2 and clathrin co-immunoprecipitate in interphase cells, an interaction that is lost in 2ME2-treated cells (Fig. 5). In contrast, the interaction between Dab2 and myosin VI is maintained in the mitotically arrested cells (Fig. 5A), stressing the specificity of the abrogation of Dab2-clathrin interactions in mitosis. The interaction of Dab2 with myosin VI is mediated through the C-terminal region of Dab2 (56Phichith D. Travaglia M. Yang Z. Liu X. Zong A.B. Safer D. Sweeney H.L. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 17320-17324Crossref PubMed Scopus (82) Google Scholar, 57Yu C. Feng W. Wei Z. Miyanoiri Y. Wen W. Zhao Y. Zhang M. Cell. 2009; 138: 537-548Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), whereas the sequence involved in binding to clathrin localizes to the central exon of Dab2 (14Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBO J. 2002; 21: 4915-4926Crossref PubMed Scopus (249) Google Scholar). Importantly, in mitosis, myosin VI also undergoes marked changes to its intracellular localization and performs important roles, related to membrane traffic in cytokinesis (58Arden S.D. Puri C. Au J.S. Kendrick-Jones J. Buss F. Mol. Biol. Cell. 2007; 18: 4750-4761Crossref PubMed Scopus (42) Google Scholar). (iv) In 2ME2-arrested cells, Dab2 is extensively phosphorylated. This phosphorylation induced a marked shift in the migration of Dab2 in SDS-PAGE, a shift that is abolished by incubation with calf intestinal phosphatase (Fig. 3D). Moreover, the mutation of Ser-393, Ser-394, and Ser-401 to alanines or the deletion of these residues reduced this shift in migration (Fig. 4B). A comparative mass spectrometry analysis of the phosphorylation of p82-WT-h in vehicle/2ME2-treated cells revealed the 2ME2-induced phosphorylation of Ser-326 (or Ser-328). Importantly, these sites, which are present in p82-5A, were not phosphorylated in this molecular context in 2ME2-arrested cells (supplemental Fig. 4). Moreover, p82-WT-h also shows significant phosphorylation on Thr(P)-Pro/Ser(P)-Pro sites (supplemental Fig. 5), suggesting that Ser-326 (or Ser-328), which is not adjacent to prolines, are not the sole sites of its mitotic phosphorylation. Furthermore, the reduction in the phosphorylation of p82-5A correlated with the decrease in its displacement from the membrane (supplemental Fig. 2) and with its continued localization to membrane-bound punctate structures in 2ME2-treated cells (Fig. 4, C and D) and in cycling cells in mitosis (supplemental Fig. 6). (v) In 2ME2-treated cells, the internalization of a receptor endowed with an FDNPXY endocytic motif is specifically inhibited (Fig. 6). Importantly, the 2ME2-induced reduction in the internalization of this receptor is dependent on the displacement of Dab2 from the membrane, as it is rescued in p82-5A-expressing cells (Fig. 6, C and D). Accordingly, we have previously shown that the internalization of this same construct is enhanced upon the expression of GFP-Dab2 in COS7 cells (49Chetrit D. Ziv N. Ehrlich M. Biochem. J. 2009; 418: 701-715Crossref PubMed Scopus (40) Google Scholar). Importantly, and similarly to what was reported by Boucrot and Kirchhausen (35Boucrot E. Kirchhausen T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7939-7944Crossref PubMed Scopus (249) Google Scholar), the internalization of an analogous TβRI-based construct endowed with a YXXΦ-like endocytic signal, which is supposedly entirely dependent on AP2, was not reduced in cells arrested in mitosis with 2ME2 (Fig. 6). Thus, arrest of cells in mitosis affects the internalization of only a subpopulation of receptors.In addition to Dab2, epsin and Eps15 are also phosphorylated in G2/M (42Chen H. Slepnev V.I. Di Fiore P.P. De Camilli P. J. Biol. Chem. 1999; 274: 3257-3260Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 59Rossé C. L'Hoste S. Offner N. Picard A. Camonis J. J. Biol. Chem. 2003; 278: 30597-30604Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 60Kariya K. Koyama S. Nakashima S. Oshiro T. Morinaka K. Kikuchi A. J. Biol. Chem. 2000; 275: 18399-18406Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Interestingly, the phosphorylation of both epsin and Eps15 also leads to a decrease in their endocytic function, through the reduction in their interaction with AP2/clathrin (42Chen H. Slepnev V.I. Di Fiore P.P. De Camilli P. J. Biol. Chem. 1999; 274: 3257-3260Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This negative regulation of endocytic proteins in mitosis contrasts with the functionality of AP2/clathrin throughout mitosis (35Boucrot E. Kirchhausen T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7939-7944Crossref PubMed Scopus (249) Google Scholar) and suggests a differential regulation of endocytic “auxiliary factors” as compared with those defined as “endocytic hubs” (according to the clathrin interactome in Ref. 61Schmid E.M. McMahon H.T. Nature. 2007; 448: 883-888Crossref PubMed Scopus (248) Google Scholar). What are putative causes and consequences of this selectivity? (i) The mitotic cell alters the extent and possibly the nature of its interactions with its neighboring cells, with the extracellular matrix, and with its microenvironment. This dynamic process may demand an alteration in the repertoire of receptors exposed at the cell surface. Selective inhibition of the internalization of a subset of receptors, in the context of a general reduction in surface area (35Boucrot E. Kirchhausen T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7939-7944Crossref PubMed Scopus (249) Google Scholar, 62Boucrot E. Kirchhausen T. PLoS ONE. 2008; 3: e1477Crossref PubMed Scopus (66) Google Scholar), may be a way of achieving this modulation. (ii) Endocytic proteins perform alternative functions in mitotic cells. Prominent examples of this phenomenon are the recruitment of clathrin to the mitotic spindle (38Yamauchi T. Ishidao T. Nomura T. Shinagawa T. Tanaka Y. Yonemura S. Ishii S. EMBO J. 2008; 27: 1852-1862Crossref PubMed Scopus (45) Google Scholar, 39Royle S.J. Bright N.A. Lagnado L. Nature. 2005; 434: 1152-1157Crossref PubMed Scopus (197) Google Scholar), the association of the endocytic adaptor ARH with centrosomal proteins (63Lehtonen S. Shah M. Nielsen R. Iino N. Ryan J.J. Zhou H. Farquhar M.G. Mol. Biol. Cell. 2008; 19: 2949-2961Crossref PubMed Scopus (33) Google Scholar), and the nonendocytic functions performed by epsin in mitosis (64Liu Z. Zheng Y. J. Cell Biol. 2009; 186: 473-480Crossref PubMed Scopus (43) Google Scholar). (iii) The mitotic cell is a different physicochemical environment than the interphase cell and may thus present different requirements for the regulation of coated pit/coated vesicle formation. For example, mitotic cells have a reduced volume (62Boucrot E. Kirchhausen T. PLoS ONE. 2008; 3: e1477Crossref PubMed Scopus (66) Google Scholar, 65Habela C.W. Sontheimer H. Cell Cycle. 2007; 6: 1613-1620Crossref PubMed Scopus (75) Google Scholar), enhanced membrane tension (36Raucher D. Sheetz M.P. J. Cell Biol. 1999; 144: 497-506Crossref PubMed Scopus (172) Google Scholar), and a different organization of the actin cytoskeleton (66Kunda P. Baum B. Trends Cell Biol. 2009; 19: 174-179Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), all of which potentially alter endocytosis. Specifically, we employed spinning disk confocal microscopy to measure the volume of ES-2 cells in metaphase and in 2ME2-mediated mitotic arrest, and we found that their volume is reduced by ∼60% in these conditions (data not shown). Such a reduction in volume would imply that the concentration of long lived proteins (the reported half-lives of clathrin and Dab2 are ∼50 and ∼16 h, respectively (10Hocevar B.A. Smine A. Xu X.X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar, 67Acton S.L. Brodsky F.M. J. Cell Biol. 1990; 111: 1419-1426Crossref PubMed Scopus (68) Google Scholar)) may be 3-fold higher under these conditions. Interestingly, the endocytic machinery is sensitive to alterations in clathrin concentration (68Moskowitz H.S. Yokoyama C.T. Ryan T.A. Mol Biol. Cell. 2005; 16: 1769-1776Crossref PubMed Scopus (37) Google Scholar). Moreover, although basic cell functions are not affected in the absence of Dab2, which is down-regulated/depleted in the initial phases of tumorigenesis of many tumor types (1Wang Z. Tseng C.P. Pong R.C. Chen H. McConnell J.D. Navone N. Hsieh J.T. J. Biol. Chem. 2002; 277: 12622-12631Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 69Karam J.A. Shariat S.F. Huang H.Y. Pong R.C. Ashfaq R. Shapiro E. Lotan Y. Sagalowsky A.I. Wu X.R. Hsieh J.T. Clin. Cancer Res. 2007; 13: 4400-4406Crossref PubMed Scopus (43) Google Scholar), an increase in the amounts of Dab2 leads to profound alterations to the organization of clathrin in cells (49Chetrit D. Ziv N. Ehrlich M. Biochem. J. 2009; 418: 701-715Crossref PubMed Scopus (40) Google Scholar, 70Mettlen M. Loerke D. Yarar D. Danuser G. Schmid S.L. J. Cell Biol. 2010; 188: 919-933Crossref PubMed Scopus (117) Google Scholar). Also, overexpression of epsin1 leads to a decrease in clathrin-mediated endocytosis (71Henne W.M. Boucrot E. Meinecke M. Evergren E. Vallis Y. Mittal R. McMahon H.T. Science. 2010; 328: 1281-1284Crossref PubMed Scopus (312) Google Scholar). Interestingly, incubation of cells in hypertonic medium (0.45 m sucrose), which reduces cell volume (by ∼50%, data not shown), induces a massive polymerization of clathrin and Dab2 into unproductive structures (49Chetrit D. Ziv N. Ehrlich M. Biochem. J. 2009; 418: 701-715Crossref PubMed Scopus (40) Google Scholar, 72Heuser J.E. Anderson R.G. J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (768) Google Scholar). Thus, a reduction in interactions with the membrane and with clathrin, through mitotic phosphorylation, may be a manner of reducing the concentration of active Dab2 and possibly also of additional auxiliary endocytic factors such as epsin and Eps15. This reduction may be part of a regulatory mechanism aimed at the continuation of endocytosis (of a subset of receptors) in mitosis, despite the broad alterations to the physicochemical environment of the cell. IntroductionDisabled-2 (Dab2) is a multidomain mitotic phosphoprotein that functions as a signal modulator and tumor suppressor (1Wang Z. Tseng C.P. Pong R.C. Chen H. McConnell J.D. Navone N. Hsieh J.T. J. Biol. Chem. 2002; 277: 12622-12631Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 2Cho S.Y. Jeon J.W. Lee S.H. Park S.S. Biochem. J. 2000; 352: 645-650Crossref PubMed Scopus (23) Google Scholar, 3Huang C.H. Cheng J.C. Chen J.C. Tseng C.P. Cell. Signal. 2007; 19: 1339-1347Crossref PubMed Scopus (22) Google Scholar, 4Xu X.X. Yi T. Tang B. Lambeth J.D. Oncogene. 1998; 16: 1561-1569Crossref PubMed Scopus (106) Google Scholar, 5Zhou J. Hsieh J.T. J. Biol. Chem. 2001; 276: 27793-27798Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Zhou J. Scholes J. Hsieh J.T. J. Biol. Chem. 2003; 278: 6936-6941Abstract Full Text Full Text PDF PubMed Scopus (58) Google" @default.
• W1979541028 created "2016-06-24" @default.
• W1979541028 creator A5012384664 @default.
• W1979541028 creator A5066054323 @default.
• W1979541028 creator A5070069763 @default.
• W1979541028 creator A5070958187 @default.
• W1979541028 creator A5080920993 @default.
• W1979541028 creator A5087218848 @default.
• W1979541028 date "2011-02-01" @default.
• W1979541028 modified "2023-10-12" @default.
• W1979541028 title "Negative Regulation of the Endocytic Adaptor Disabled-2 (Dab2) in Mitosis" @default.
• W1979541028 cites W1526105662 @default.
• W1979541028 cites W1683260590 @default.
• W1979541028 cites W1910209889 @default.
• W1979541028 cites W1963827447 @default.
• W1979541028 cites W1965232742 @default.
• W1979541028 cites W1970268623 @default.
• W1979541028 cites W1973744676 @default.
• W1979541028 cites W1975224427 @default.
• W1979541028 cites W1975235201 @default.
• W1979541028 cites W1981484848 @default.
• W1979541028 cites W1982610759 @default.
• W1979541028 cites W1983921682 @default.
• W1979541028 cites W1988616931 @default.
• W1979541028 cites W1989319811 @default.
• W1979541028 cites W1990781591 @default.
• W1979541028 cites W1990883677 @default.
• W1979541028 cites W1991418321 @default.
• W1979541028 cites W1993169452 @default.
• W1979541028 cites W1993450321 @default.
• W1979541028 cites W1994912740 @default.
• W1979541028 cites W1997140657 @default.
• W1979541028 cites W2002390599 @default.
• W1979541028 cites W2005841227 @default.
• W1979541028 cites W2017201573 @default.
• W1979541028 cites W2020952581 @default.
• W1979541028 cites W2029241563 @default.
• W1979541028 cites W2033344378 @default.
• W1979541028 cites W2037293612 @default.
• W1979541028 cites W2037471767 @default.
• W1979541028 cites W2038867812 @default.
• W1979541028 cites W2041848251 @default.
• W1979541028 cites W2042608426 @default.
• W1979541028 cites W2043779556 @default.
• W1979541028 cites W2045407209 @default.
• W1979541028 cites W2048404650 @default.
• W1979541028 cites W2048938594 @default.
• W1979541028 cites W2053804215 @default.
• W1979541028 cites W2056238689 @default.
• W1979541028 cites W2057862712 @default.
• W1979541028 cites W2059697575 @default.
• W1979541028 cites W2062706888 @default.
• W1979541028 cites W2068972095 @default.
• W1979541028 cites W2077569426 @default.
• W1979541028 cites W2079290695 @default.
• W1979541028 cites W2082220504 @default.
• W1979541028 cites W2085105723 @default.
• W1979541028 cites W2087008002 @default.
• W1979541028 cites W2092250528 @default.
• W1979541028 cites W2096154351 @default.
• W1979541028 cites W2096892171 @default.
• W1979541028 cites W2101067824 @default.
• W1979541028 cites W2101636340 @default.
• W1979541028 cites W2108098596 @default.
• W1979541028 cites W2113088261 @default.
• W1979541028 cites W2115994196 @default.
• W1979541028 cites W2124733101 @default.
• W1979541028 cites W2124846937 @default.
• W1979541028 cites W2129697908 @default.
• W1979541028 cites W2129777487 @default.
• W1979541028 cites W2134148250 @default.
• W1979541028 cites W2134832683 @default.
• W1979541028 cites W2139362624 @default.
• W1979541028 cites W2140638448 @default.
• W1979541028 cites W2141396232 @default.
• W1979541028 cites W2144007269 @default.
• W1979541028 cites W2145295859 @default.
• W1979541028 cites W2149263983 @default.
• W1979541028 cites W2161899008 @default.
• W1979541028 cites W2166304697 @default.
• W1979541028 cites W2254508525 @default.
• W1979541028 cites W2404021914 @default.
• W1979541028 cites W282408398 @default.
• W1979541028 doi "https://doi.org/10.1074/jbc.m110.161851" @default.
• W1979541028 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3037652" @default.
• W1979541028 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21097498" @default.
• W1979541028 hasPublicationYear "2011" @default.
• W1979541028 type Work @default.
• W1979541028 sameAs 1979541028 @default.
• W1979541028 citedByCount "27" @default.
• W1979541028 countsByYear W19795410282012 @default.
• W1979541028 countsByYear W19795410282013 @default.
• W1979541028 countsByYear W19795410282014 @default.
• W1979541028 countsByYear W19795410282015 @default.
• W1979541028 countsByYear W19795410282016 @default.
• W1979541028 countsByYear W19795410282017 @default.
• W1979541028 countsByYear W19795410282018 @default.
• W1979541028 countsByYear W19795410282019 @default.

• next