Signaling from Rho to the Actin Cytoskeleton Through Protein Kinases ROCK and LIM-kinase
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After LPA stimulation N1E-115 cells were lysed with 10 mM tris-HCl (pH 7.5) containing 1 mM EGTA 5 mM MgCl 2 1 mM dithiothreitol 1% (v/v) Triton X-100 and mixtures of phosphatase inhibitors and protease inhibitors. Lysates were centrifuged and the supernatant corresponding to 2 × 10 4 cells was subjected to two-dimensional electrophoresis with a linear pH gradient from 3.5 to 10 followed by autoradiography.
Purification and identification details of P1 and P2 proteins are available at www.sciencemag.org/feature/data/1041678.shl.
In the transfection experiments in this study COS-7 cells were transfected with combinations of the following vectors: pFL-C1-FLAG–tagged wild-type (WT) or S3A cofilin pCAG-Myc–tagged ROCKΔ1 (14) pUCD2-SRα-FLAG–tagged LIMK1 (17) pUC-SRα-Myc–tagged LIMK 2 [
] pCMV5-FLAG–tagged N 17 -Rac (8) pEXV-Myc–tagged V 14 -Rho (14) and pCAG-Myc–tagged ROCK-KDIA (14). After transfection cells were cultured for 15 hours in Dulbecco's minimum essential medium containing 10% fetal bovine serum (Fig. 1C) or in serum-free OPTI-MEM (Gibco) (Figs. 2B and 3A). In the experiments in Figs. 1C and 3A the cells were then labeled for 4 hours with [ 32 P]orthophosphate (50 μCi/ml). Cells were lysed in 20 mM tris-HCl (pH 7.5) containing 1 mM EDTA 1 mM EGTA 5 mM MgCl 2 5 mM 2-mercaptoethanol 0.05% Triton X-100 and mixtures of phosphatase inhibitors and protease inhibitors. FLAG-tagged cofilin was immunoprecipitated with anti-FLAG (M2) affinity beads (Sigma) and subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography or immunoblot analysis (Figs. 1C and 3A). FLAG-tagged LIMK1 was immunoprecipitated and subjected to kinase assay (16) (Fig. 2B).
His 6 -tagged cofilin was expressed in Escherichia coli and His 6 -tagged ROCKΔ4 (residues 1 to 477) was produced in Sf9 cells. Kinase reactions were done in a total volume of 23 μl at 30°C for 10 min with 50 μM [γ- 32 P]ATP (adenosine 5′-triphosphate) and 4 μg of either cofilin or histone type 2 as substrates in kinase buffer [25 mM Hepes - NaOH (pH 7.5) 10 mM MgCl 2 5 mM MnCl 2 0.1 mM EGTA 0.05% Brij 35 5 mM 2-mercaptoethanol phosphatase inhibitors and protease inhibitors].
; N. Yang et al. ibid. p. 809.
M. Maekawa et al. unpublished data.
N. Watanabe et al. EMBO J. 16 1307 (1997).
Supported by grants from the Ministry of Education Science and Culture of Japan and from the Human Frontier Science Program. We thank E. Nishida I. Yahara and T. Doi for advice and A. Shinohara for mass spectrometry. M.M. thanks S. Maekawa for encouragement.