Table S1 shows the levels of HUTS-21 epitope on cells transfected with various forms of DAP-kinase. Fig. S1 shows that DAP-kinase does not affect cell surface
expression of integrins.
Acknowledgements
We thank Gena Whitney for CD2-FAK, Caroline Damsky for AIIB2 antibody, Lillian Shum for p53 reporter, Hong-Chen Chen for discussion and Randall Kramer for critical reading the manuscript. This work was supported by NSC Frontier Grant 90-2321-B-002-004 to R.-H. C, and Grants NSC 89-2134-B-002-349 and NTUH 91-N020 to C.-C. Yao.
Abbreviations
DAP-kinase, death associated protein kinase DMEM, Dulbecco’s modified Eagle’s medium ECM, extracellular matrix
EGF, epidermal growth factor FCS, fetal calf serum
MLC, regulatory light chain of myosin II PBS, phosphate buffered saline
Poly-HEMA, polyhydroxyethylmethacrylate
References
Almeida, E.A., D. Ilic, Q. Han, C.R. Hauck, F. Jin, H. Kawakatsu, D.D.
Schlaepfer and C.H. Damsky. 2000. Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase. J Cell Biol 149: 741-54.
Bodary, S.C. and J.W. McLean. 1990. The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line. J Biol Chem 265: 5938-41.
Chan, P.Y., S.B. Kanner, G. Whitney and A. Aruffo. 1994. A
transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK. J Biol Chem 269:
20567-74.
Cohen, O., E. Feinstein and A. Kimchi. 1997. DAP-kinase is a
Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. Embo J 16: 998-1008.
Cohen, O., B. Inbal, J.L. Kissil, T. Raveh, H. Berissi, T. Spivak-Kroizaman, E.
Feinstein and A. Kimchi. 1999. DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain. J Cell Biol 146:
141-8.
Cohen, O. and A. Kimchi. 2001. DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer. Cell Death Differ 8: 6-15.
Deiss, L.P., E. Feinstein, H. Berissi, O. Cohen and A. Kimchi. 1995.
Identification of a novel serine/threonine kinase and a novel 15-kD protein as
potential mediators of the gamma interferon-induced cell death. Genes Dev 9: 15-30.
Frisch, S.M. and E. Ruoslahti. 1997. Integrins and anoikis. Curr Opin Cell Biol 9:
701-6.
Frisch, S.M. and R.A. Screaton. 2001. Anoikis mechanisms. Curr Opin Cell Biol 13: 555-62.
Frisch, S.M., K. Vuori, E. Ruoslahti and P.Y. Chan-Hui. 1996. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134: 793-9.
Gailit, J. and E. Ruoslahti. 1988. Regulation of the fibronectin receptor affinity by divalent cations. J Biol Chem 263: 12927-32.
Giancotti, F.G. and E. Ruoslahti. 1999. Integrin signaling. Science 285: 1028-32.
Ginsberg, M.H., X. Du and E.F. Plow. 1992. Inside-out integrin signalling. Curr Opin Cell Biol 4: 766-71.
Hughes, P.E. and M. Pfaff. 1998. Integrin affinity modulation. Trends Cell Biol 8:
359-64.
Hungerford, J.E., M.T. Compton, M.L. Matter, B.G. Hoffstrom and C.A. Otey.
1996. Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J Cell Biol 135: 1383-90.
Ilic, D., E.A. Almeida, D.D. Schlaepfer, P. Dazin, S. Aizawa and C.H. Damsky.
1998. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol 143: 547-60.
Ilic, D., C.H. Damsky and T. Yamamoto. 1997. Focal adhesion kinase: at the crossroads of signal transduction. J Cell Sci 110: 401-7.
Inbal, B., O. Cohen, S. Polak-Charcon, J. Kopolovic, E. Vadai, L. Eisenbach and A. Kimchi. 1997. DAP kinase links the control of apoptosis to metastasis. Nature 390:
180-4.
Inbal, B., G. Shani, O. Cohen, J.L. Kissil and A. Kimchi. 2000. Death-associated protein kinase-related protein 1, a novel serine/threonine kinase involved in apoptosis.
Mol Cell Biol 20: 1044-54.
Inbal, B., S. Bialik, I. Sabanay, G. Shani and A. Kimchi. 2002. DAP kinase and
DRP-1 mediated membrane blebbing and the formation of autophagic vesicles during programmed cell death. J Cell Biol 157: 455-68.
Jang, C.W., C.H. Chen, C.C. Chen, J.Y. Chen, Y.H. Su and R.H. Chen. 2002.
TGF-beta induces apoptosis through Smad-mediated expression of DAP- kinase. Nat Cell Biol 4: 51-8.
Jost, M., T.M. Huggett, C. Kari and U. Rodeck. 2001. Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway. Mol Biol Cell 12: 1519-27.
Kawai, T., M. Matsumoto, K. Takeda, H. Sanjo and S. Akira. 1998. ZIP kinase, a novel serine/threonine kinase which mediates apoptosis. Mol Cell Biol 18: 1642-51.
Li, G., R. Fridman and H.R. Kim. 1999. Tissue inhibitor of metalloproteinase-1 inhibits apoptosis of human breast epithelial cells. Cancer Res 59: 6267-75.
Marti, A., Z. Luo, C. Cunningham, Y. Ohta, J. Hartwig, T.P. Stossel, J.M.
Kyriakis and J. Avruch. 1997. Actin-binding protein-280 binds the stress-activated protein kinase (SAPK) activator SEK-1 and is required for tumor necrosis
factor-alpha activation of SAPK in melanoma cells. J Biol Chem 272: 2620-8.
Mould, A.P., S.K. Akiyama and M.J. Humphries. 1995. Regulation of integrin alpha 5 beta 1-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+. J Biol Chem 270: 26270-7.
Ni, H., A. Li, N. Simonsen and J.A. Wilkins. 1998. Integrin activation by dithiothreitol or Mn2+ induces a ligand-occupied conformation and exposure of a novel NH2-terminal regulatory site on the beta1 integrin chain. J Biol Chem 273:
7981-7.
O'Toole, T.E., Y. Katagiri, R.J. Faull, K. Peter, R. Tamura, V. Quaranta, J.C.
Loftus, S.J. Shattil and M.H. Ginsberg. 1994. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol 124: 1047-59.
Otto, I.M., T. Raabe, U.E. Rennefahrt, P. Bork, U.R. Rapp and E. Kerkhoff. 2000.
The p150-Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization. Curr Biol 10: 345-8.
Pelled, D., T. Raveh, C. Riebeling, M. Fridkin, H. Berissi, A.H. Futerman and A.
Kimchi. 2001. Death-associated protein- (DAP) kinase plays a central role in ceramide- induced apoptosis in cultured hippocampal neurons. J Biol Chem 277:
1957-61.
Puthalakath, H., D.C. Huang, L.A. O'Reilly, S.M. King and A. Strasser. 1999.
The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex. Mol Cell 3: 287-96.
Puthalakath, H., A. Villunger, L.A. O'Reilly, J.G. Beaumont, L. Coultas, R.E.
Cheney, D.C. Huang and A. Strasser. 2001. Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis.
Science 293: 1829-32.
Raveh, T., G. Droguett, M.S. Horwitz, R.A. DePinho and A. Kimchi. 2001. DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol 3: 1-7.
Raveh, T. and A. Kimchi. 2001. DAP kinase-a proapoptotic gene that functions as a tumor suppressor. Exp Cell Res 264: 185-92.
Sanjo, H., T. Kawai and S. Akira. 1998. DRAKs, novel serine/threonine kinases related to death-associated protein kinase that trigger apoptosis. J Biol Chem 273:
29066-71.
Schoenwaelder, S.M. and K. Burridge. 1999. Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol 11: 274-86.
Takagi, J., H.P. Erickson and T.A. Springer. 2001. C-terminal opening mimics 'inside-out' activation of integrin alpha5beta1. Nat Struct Biol 8: 412-6.
Tsai, Y.T., Y.H. Su, S.S. Fang, T.N. Huang, Y. Qiu, Y.S. Jou, H.M. Shih, H.J.
Kung and R.H. Chen. 2000. Etk, a Btk family tyrosine kinase, mediates cellular transformation by linking Src to STAT3 activation. Mol Cell Biol 20: 2043-54.
Turner, C.E. 2000. Paxillin interactions. J Cell Sci 113 Pt 23: 4139-40.
Valentinis, B., K. Reiss and R. Baserga. 1998. Insulin-like growth factor-I-mediated survival from anoikis: role of cell aggregation and focal adhesion kinase. J Cell Physiol 176: 648-57.
van de Wiel-van Kemenade, E., Y. van Kooyk, A.J. de Boer, R.J. Huijbens, P.
Weder, W. van de Kasteele, C.J. Melief and C.G. Figdor. 1992. Adhesion of T and B lymphocytes to extracellular matrix and endothelial cells can be regulated through the beta subunit of VLA. J Cell Biol 117: 461-70.
van Kooyk, Y. and C.G. Figdor. 2000. Avidity regulation of integrins: the driving force in leukocyte adhesion. Curr Opin Cell Biol 12: 542-7.
Vinogradova, O., T. Haas, E.F. Plow and J. Qin. 2000. A structural basis for integrin activation by the cytoplasmic tail of the alpha IIb-subunit. Proc Natl Acad Sci U S A 97: 1450-5.
Xu, L.H., X. Yang, C.A. Bradham, D.A. Brenner, A.S. Baldwin, Jr., R.J. Craven and W.G. Cance. 2000. The focal adhesion kinase suppresses
transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways. J Biol Chem 275:
30597-604.
Yamamoto, M., T. Hioki, T. Ishii, S. Nakajima-Iijima and S. Uchino. 2002. DAP kinase activity is critical for C2-ceramide-induced apoptosis in PC12 cells. Eur J Biochem 269: 139-147.
Figure Legends
Fig. 1. DAP-kinase induces apoptosis-independent morphological changes in 293T cells. (A) 293T cells were transiently transfected with expression vectors for various DAP-kinase proteins or the vector backbone. The transfection efficiency was 90 %.
Expression of the corresponding proteins was determined by Western blot analysis.
(B) Morphologies of the 293T transfectants. Two days after transfection, cell morphology was examined by phase-contrast microscopy and the percentage of cells with round morphology is indicated as mean ± S.D. Bar, 10 M. (C, D, and E) Apoptotic analyses of the 293T transfectants. Two days after transfection, cells as described in (A) were harvested and analyzed for their sub-G1 DNA content by flow cytometry analysis (C), DNA fragmentation by Cell-Death Detection ELISA (D), and binding to Annexin V (E). As a control, 293T cells were irradiated with UV at 0.04 J/cm2 followed by 24 h of incubation, and then subjected to the same analyses.
Fig. 2. DAP-kinase inhibits integrin-mediated cell adhesion and ECM signal
transduction. (A and B) 293T cells were transfected with various expression vectors as in Fig. 1. Immediately after transfection, cells were incubated with or without 50
M of zVAD-FMK throughout the culture and assay periods. Two days
post-transfection, cells were replated on fibronectin (A), or laminin (B) and allowed to adhere for 40 or 25 min as indicated. The percentage of adhesion was calculated as described in Materials and Methods. Data from three separate experiments are presented as mean ± S.D. (C) Cells as in (A) were assayed for their attachment to poly-L-lysine. Adhesion assays were performed as in (A). (D) Effect of
DAP-kinase on tyrosine phosphorylation of FAK and paxillin. 293T transfectants as described in (A) were plated on fibronectin for 1 h. Cells in suspension and attached
were combined and then lysed. FAK or paxillin tyrosine phosphorylation was detected by immunoprecipitations with anti-FAK or anti-paxillin antibody as indicated, followed by Western blotting with antibody to phosphotyrosine.
Fig. 3. DAP-kinase suppresses integrins activity. (A and B) Activation of
1-integrin abrogates the anti-adhesion effect of DAP-kinase. 293T cells transfected with DAP-kinase, ΔCaM or vector control were detached at 2 days after transfection.
Cells were incubated with the TS2/16 anti-1 activating antibody (A), or MnCl2 (B) and then assayed for their attachment to fibronectin. (C) Effect of DAP-kinase on the expression of the active conformation of 1 integrin. 293T transfectants were incubated with antibody B44 specific to the activated 1 integrin or buffer alone (control), followed by labeling with the FITC-conjugated secondary antibody.
Alternatively, cells were pre-incubated with 2 mM MnCl2 for 20 min at 37°C (Mn), and then subjected to the same immunostaining. Fluorescence intensity was
determined by FACS analysis, and results of a representative experiment are shown.
Mean fluorescence intensity (± S.D.) calculated from three independent experiments is listed on the right (*, P < 0.05; **, P < 0.005 compared with the vector control without receiving Mn).
Fig. 4. DAP-kinase inhibits adhesion of NIH3T3 cells by inside-out modulation of integrin activity. (A) NIH3T3 cells were infected with retroviruses carrying various DAP-kinase or the control virus and expression of the corresponding DAP-kinase proteins was determined at 4 days post-infection by Western blot analysis. (B) Cells as in (A) were assayed for their sub-G1 DNA content. As a control, NIH3T3 cells were irradiated with UV at 0.02 J/cm2 followed by 24 h incubation. (C) Cells as described in (A) were assayed for their attachment to fibronectin. Cells were
allowed to adhere for 40 or 25 min and adhesion assays were performed as described in Fig. 2. (D) Mn2+ treatment abrogates the inhibitory role of DAP-kinase in cell adhesion. Cells as in (A) were pre-incubated with or without MnCl2 before assaying for their attachment to fibronectin.
Fig. 5. DAP-kinase promotes apoptosis by blocking ECM survival signals. (A) DAP-kinase induces apoptosis in cells plated on matrix. NIH3T3 cells infected with various retroviruses at 4 days post-infection were plated on fibronectin (FN) or poly-L-lysine (PLL) and cultured in the absence of serum for 12 h. Cells were harvested and apoptotic cells were determined by Cell-Death Detection ELISA.
Data from triplicate experiments are presented as mean ± S.D. (B) NIH3T3 cells infected, plated and cultured as in (A) were subjected to caspase 3 activity assays.
(C) NIH3T3 cells were transiently transfected with the combinations of expression vectors as indicated. The transfection efficiency was around 50%. Expression of various proteins was assayed by Western blot analyses with antibodies to DAP-kinase (upper panel), FAK (middle panel) and tubulin (bottom panel). (D) CD2-FAK prevents DAP-kinase-induced apoptosis. NIH3T3 transfectants as in (C) were plated on fibronectin and cultured for 12 h. under serum-starved conditions. Apoptotic cells were assayed as described in (A).
Fig. 6. Activation of integrin restores FAK tyrosine phosphorylation and protects cells from DAP-kinase-induced apoptosis. (A). NIH3T3 cells were transfected with various DAP-kinase expression constructs as indicated. Thirty-six hours after transfection, cells were cultured in serum-free medium for 8 h. The cells were then detached, incubated with or without 5 g/ml of 9EG7 in DMEM containing 1% BSA for 30 min at 37ºC, and plated onto fibronectin for 1 h. Cells were then harvested,
lysed and subjected to Western blot analysis with antibodies to FAK (lower panel) and FAK phosphorylated at tyrosine 397 (upper panel). (B and C) NIH3T3 cells
transfected, cultured as in (A) were incubated with the activating antibody 9EG7 (B) or the non-activating antibody MB1.2 (C). Cells were then plated on fibronectin and cultured in serum-starved conditions in the presence of the corresponding antibody for 7 h. Apoptotic cells were quantitated as described in Fig. 5.
Fig. 7. DAP-kinase induces an anoikis-like apoptosis in epithelial cells. (A) MCF10A cells were infected with various DAP-kinase expressing retroviruses or the control virus. Infected cells were selected by puromycin and then harvested at 4 days post-infection. Expression of various forms of DAP-kinase was detected by Western blot analysis. (B) Activation of 1 integrin rescues the survival of
DAP-kinase- or ΔCaM-expressing cells. MCF10A derivatives as in (A) were plated on fibronectin or poly-HEMA and cultured in EGF-deprived medium with or without TS2/16 for 24 h. Apoptotic cells were quantitated as described in Fig. 5.
Fig. 8. DAP-kinase can no longer promote apoptosis in cells resistant to anoikis.
(A) Expression of the various forms of DAP-kinase in BT474 breast carcinoma cells.
BT474 cells were transiently transfected with expression vectors for various
DAP-kinase proteins or a control vector. The transfection efficiency was around 60
%. Two days after transfection, cells were lysed and subjected to Western blot analyses with antibodies as indicated. (B) BT474 transfectants as in (A) were plated on poly-HEMA or fibronectin and cultured for 24 h under serum-free conditions.
Apoptotic cells were assayed as described in Fig. 5.
Fig. 9. Integrin or FAK activation blocks the induction of p53 by DAP-kinase. (A)
NIH3T3 cells were co-transfected with p53-TA-luc and pRK5-gal, in the presence or absence of various DAP-kinase expression constructs and CD2-FAK. At 36 h post-transfection, cells were plated on fibronectin and cultured in serum-free medium for 12 h. and then lysed for measuring reporter activities. Values are mean ± S.D. of triplicate assays. (B) NIH3T3 cells were co-transfected with p53-TA-luc and
pRK5-gal, in the presence or absence of various DAP-kinase expression constructs.
Cells were then serum-starved, incubated with or without 9EG7, and plated on fibronectin as described in Materials and Methods. Reporter activities were
determined as in (A). (C) NIH3T3 cells transfected as indicated were cultured for 36 h in growth medium and then plated on fibronectin-coated dishes. Cells were
cultured under serum-free conditions for 6 h and subjected to Western blot analysis with antibodies as indicated. (D) A model illustrating the mechanism by which DAP-kinase induces apoptosis (see text).