Inhibition of myosin light chain kinase by p21-activated kinase

Science

Volumn 283, Issue 5410, 1999, Pages 2083-2085

  Sanders, Luraynne C ^a   Matsumura, Fumio ^b   Bokoch, Gary M ^a   De Lanerolle, Primal ^c  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

^b RUTGERS UNIVERSITY   (United States)

^c UNIVERSITY OF ILLINOIS AT CHICAGO   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ACTIN; GUANINE NUCLEOTIDE BINDING PROTEIN; GUANOSINE TRIPHOSPHATASE; MYOSIN; MYOSIN LIGHT CHAIN KINASE; PROTEIN CDC42; PROTEIN P21; RAC PROTEIN; RHO FACTOR; UNCLASSIFIED DRUG;

ANIMAL CELL; ARTICLE; CELL ADHESION; CELL SPREADING; CONTROLLED STUDY; CYTOSKELETON; ENZYME ACTIVITY; ENZYME INHIBITION; ENZYME PHOSPHORYLATION; EXTRACELLULAR MATRIX; HAMSTER; HUMAN; HUMAN CELL; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION;

ANIMALS; CDC42 GTP-BINDING PROTEIN; CELL ADHESION; CELL CYCLE PROTEINS; CELL LINE; CELL MOVEMENT; CELL SIZE; CRICETINAE; CYTOSKELETON; DIACETYL; GTP PHOSPHOHYDROLASES; GTP-BINDING PROTEINS; HELA CELLS; HUMANS; INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS; MYOSIN LIGHT CHAINS; MYOSIN-LIGHT-CHAIN KINASE; MYOSINS; PHOSPHORYLATION; PHOSPHOSERINE; PROTEIN-SERINE-THREONINE KINASES; RAC GTP-BINDING PROTEINS; SIGNAL TRANSDUCTION;

ANIMALIA; CRICETINAE;

EID: 0033605738     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5410.2083     Document Type: Article

References (33)

1. A. K. Wilson et al., Cancer Metastasis Rev. 11, 79 (1992).
2. T. J. Mitchison and L. P. Cramer, Cell 84, 371 (1996).
3. K. Burridge and M. Chrzanowska-Wodnicka, Annu. Rev. Cell Dev. Biol. 12, 463 (1996).
4. C. D. Nobes and A. Hall, Cell 81, 53 (1995).
5. A. Hall, Science 279, 509 (1998).
6. S. Dharmawardhane et al., J. Cell Biol. 138, 1265 (1997).
7. M. A. Sells et al., Curr. Biol. 7, 202 (1997).
8. R. H. Daniels; P. S. Hall, G. M. Bokoch, EMBO J. 17, 754 (1998).
9. R. L. Debiasio et al., J. Cell Biol. 107, 2631 (1988).
10. A. K. Wilson et al., ibid. 114, 277 (1991).
11. J. Howard, Nature 389, 561 (1997).
12. P. de Lanerolle and R. J. Paul, Am. J. Physiol. 261, L1 (1991).
13. A. P. Somlyo and A. V. Somlyo, Nature 372, 231 (1994).
14. R. L. Klemke et al., J. Cell Biol. 137, 481 (1997).
15. P. de Lanerolle, M. Nishikawa, D. A. Yost, R. S. Adelstein, Science 223, 1415 (1984).
16. M. G. Tansey et al., J. Biol. Chem. 267, 12511 (1992).
17. P. de Lanerolle et al., in preparation.
18. -7 M calmodulin, 5 mM DTT, and purified regulatory MLC (10 μg). Aliquots were removed at various times and subjected to SDS-PAGE. The bands representing MLC were excised and counted. To quantify changes in MLCK activity in cells, we immunoprecipitated MLCK from nontransfected HeLa cells or from HeLa cells expressing LacZ, wtPAK1, or PAK1 (T423E) as described [P. de Lanerolle, G. Gorgas, X. Li, K. Schluns, J. Biol. Chem. 268, 16883 (1993)] with affinity-purified goat antibodies to MLCK [P. de Lanerolle et al., Circ. Res. 68, 457 (1991)] and protein A-Sepharose. The beads were washed extensively and then assayed for MLCK activity as described. HeLa cells were used in these experiments because the MLCK antibody reacts poorly with BHK-21 MLCK on protein immunoblot analysis.
19. -7 M calmodulin, 5 mM DTT, and purified regulatory MLC (10 μg). Aliquots were removed at various times and subjected to SDS-PAGE. The bands representing MLC were excised and counted. To quantify changes in MLCK activity in cells, we immunoprecipitated MLCK from nontransfected HeLa cells or from HeLa cells expressing LacZ, wtPAK1, or PAK1 (T423E) as described [P. de Lanerolle, G. Gorgas, X. Li, K. Schluns, J. Biol. Chem. 268, 16883 (1993)] with affinity-purified goat antibodies to MLCK [P. de Lanerolle et al., Circ. Res. 68, 457 (1991)] and protein A-Sepharose. The beads were washed extensively and then assayed for MLCK activity as described. HeLa cells were used in these experiments because the MLCK antibody reacts poorly with BHK-21 MLCK on protein immunoblot analysis.
20. -7 M calmodulin, 5 mM DTT, and purified regulatory MLC (10 μg). Aliquots were removed at various times and subjected to SDS-PAGE. The bands representing MLC were excised and counted. To quantify changes in MLCK activity in cells, we immunoprecipitated MLCK from nontransfected HeLa cells or from HeLa cells expressing LacZ, wtPAK1, or PAK1 (T423E) as described [P. de Lanerolle, G. Gorgas, X. Li, K. Schluns, J. Biol. Chem. 268, 16883 (1993)] with affinity-purified goat antibodies to MLCK [P. de Lanerolle et al., Circ. Res. 68, 457 (1991)] and protein A-Sepharose. The beads were washed extensively and then assayed for MLCK activity as described. HeLa cells were used in these experiments because the MLCK antibody reacts poorly with BHK-21 MLCK on protein immunoblot analysis.
21. L. S. Price, J. Leng, M. A. Schwartz, G. M. Bokoch, Mol. Biol. Cell 9, 1863 (1998).
22. 7 plaque-forming units per milliliter. Viral stocks were stored at -80°C. The virus was activated per manufacturer's instructions, and BHK-21 and HeLa cells were infected in serum-free medium. Transfection efficiency of recombinant virus was routinely >95% in BHK-21 cells and >50% in HeLa cells. Cells were allowed to express protein for 6 to 8 hours after infection in serum-free medium before use in experiments.
23. For cell adhesion assays and immunofluorescence, cells were suspended in basal medium (Glasco minimal essential medium, Life Technologies) containing no serum and seeded in six-well plastic plates containing coverslips coated with fibronectin (20 μg/ml). Cells attached to coverslips were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min, and then incubated with antibody to Myc (9E10) at 1:300 or with antibody to β-galactosidase (β-Gal) at 1:5000 (Promega, Madison, WI) for 1 hour. Cells were then incubated for 1 hour with rhodamine phalloidin 1:500 (Sigma) or with fluorescein isothiocyanate-conjugated antibody to mouse immunoglobulin G (IgG) 1:300 (Cappel Labs, Cochranville, PA) or with both, washed with phosphate-buffered saline, and mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). Slides were examined with a Nikon Labophot-ZDFX-DX epifluorescence microscope, and images were photographed with a 35-mm camera and Kodak T-MAX film. For inhibition studies, various concentrations of BDM (Sigma) and ML-7 (Calbiochem, La Jolla, CA) were added to the culture medium.
24. L. P. Cramer and T. J. Mitchison, J. Cell Biol. 131, 179 (1995).
25. Y. Yamakita, S. Yamashiro, F. Matsumura, ibid. 124, 129 (1994).
26. L. C. Sanders and G. M. Bokoch, unpublished data.
27. M. Saitoh, T. Ishikawa, S. Matsushima, M. Naka, H. Hidaka, J. Biol. Chem. 262, 7801 (1997).
28. 19 [F. Matsumura et al., J. Cell Biol. 140, 119 (1998)]. Protein bands were visualized with horseradish peroxidase-conjugated goat antibody to rabbit IgG (Pierce, Rockford, IL) and with chemiluminescence.
29. M. Chrzanowska-Wodnicka and K. Burridge, J. Cell Biol. 133, 1403 (1996).
30. K. Kimura et al., Science 273, 245 (1996).
31. M. Amano et al., J. Biol. Chem. 271, 29246 (1996).
32. F. Matsumura and G. M. Bokoch, unpublished data.
33. Supported by a U.S. Army Research Breast Cancer Program award to L.C.S. and by grants from the U.S. Public Health Service to F.M., to G.M.B., and to P.d.L. This is publication 11936-IMM from The Scripps Research Institute.