CaMK-II promotes focal adhesion turnover and cell motility by inducing tyrosine dephosphorylation of FAK and paxillin.

NIH/3T3 Culture and Harvesting NIH/3T3 cells were used in all studies and were maintained on tissue culture dishes (Nunc, Rochester, NY) at 37°C in DMEM with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). Cells were sub-cultured every 3–4 days, never exceeding 95% confluency. When specified, dishes were pre-incubated with 1 μg/ml human fibronectin (Invitrogen) in PBS (Phosphate Buffered Saline) for 1 h at 37°C or overnight at 4°C, washed once with PBS and then placed into DMEM/10% FBS just prior to plating cells. Cells were harvested by trypsinization, washed in ice-cold PBS and then resuspended in homogenization buffer, which consisted of 30 mM Hepes pH 7.4, 20 mM MgCl 2 , 80 mM β-glycerol phosphate, 2.6 mM EGTA, 0.1 μM okadaic acid, 1 μg/ml each chymostatin, leupeptin, antipain, pepstatin and soybean trypsin inhibitor. Cells were lysed using two 4-s bursts from a probe sonicator (Misonix, Farmingdale, NY) and then centrifuged at 12,000 g for 15 min at 4°C.

Plasmid Constructs EGFP-linked

CaMK-II constructs used in this study were prepared as previously described [ Lantsman and Tombes, 2005 ]. The δ C CaMK-II variant used here represents the simplest splice variant and the most common form expressed in these cells [ Tombes et al., 2003 ]. EGFP-paxillin, dsRed-paxillin and EGFP-FAK were prepared as described [ Webb et al., 2004 ; Brown et al., 2006 ]. EGFP-talin and vinculin were generous gifts from Dr. Kenneth Yamada, National Institutes of Health, Bethesda, MD and Dr. Benjamin Geiger, Weizmann Institute of Science, Rehovot Israel, respectively.

Transfection and Microscopy

Freshly sub-cultured cells were transfected with Lipofectamine 2000 as specified (Invitrogen). Typically, transfections utilized 20 μg of total DNA for 100-mm plates and 4 μg for 6-well dishes. Co-transfections used equal amounts of each construct. Live or formaldehyde fixed cells were imaged in phase contrast, traditional fluorescence (Fm) or Total Internal Reflection Fluorescence microscopy (TIRFm) using an IX-70 inverted microscope equipped with a 12-bit black/white F-View CCD camera and processed using Microsuite-B3SV Version 3.2 software (Olympus, Melville, NY).

TIRF

Illumination utilized a 10 mW Argon-ion laser (Melles Griot, Carlsbad, CA) for 488 nm illumination via a 60×/1.45NA PlanApo objective (Olympus). Living cells were maintained at 37°C using a stage heater (20/20 Technology, Wilmington, NC) Analysis of Cell Migration, Spreading and Focal Adhesion Size NIH/3T3 cells were cultured just to confluency and then scratch-wounded using a single stroke from a fine pipette tip. Cells were immediately re-fed with fresh 10% FBS/DMEM containing either vehicle alone, 10 μM KN-93 or 20 μM myr-AIP. Cells were then imaged under phase contrast at 0, 3 and 6 h post wounding. The microscope stage was maintained at 37°C. Between imaging sessions, cells were returned to the incubator. Lamellipodial dynamics were captured over 10 min using phase contrast imaging and Olympus Microsuite-B3SV Version 3.2 software (Olympus) for cells plated on FN in the presence of vehicle control or 20 μM myr-AIP ( supplemental videos 1 and 2 ). Cell migration rates were calculated from images taken 1 and 4 h after plating on FN with Microsuite-B3SV Version 3.2. Diameter measurements for cell spreading and focal adhesion size analysis were also computed with Microsuite-B3SV Version 3.2 software. Minimum and maximum threshold values for focal adhesion size were 1.7 and 35 μm 2 , respectively. T-tests were employed to determine statistically significant differences, as indicated by P -value thresholds. Immunoblotting Transfected NIH/3T3 cells were harvested as described above (see Cell Culture and Harvesting ). Samples were subjected to SDS-PAGE on 4–15 or 10% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to 0.45-μm nitrocellulose sheets, and then blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.01% Tween-20 and 0.1% sodium azide (TBSTA) and 5% pre-immune goat serum. Blots were then incubated overnight with 1 μg/ml primary antibody in 5% BSA, TBSTA. After a 2-h incubation with 2 μg/ ml alkaline phosphatase conjugated goat anti-mouse or rabbit IgG, blots were developed with 0.25 mg/ml BCIP/ NBT (Roche, Indianapolis, IN) in phosphatase buffer, pH 9.4 [ Lantsman and Tombes, 2005 ]. Reagents Total FAK and paxillin antibodies were obtained from BD Biosciences (Rockville, MD), phospho-Y 31 paxillin and phospho-S 843 FAK antibodies from Biosource/Invitrogen, and phospho-Y 925 FAK antibody from Cell Signaling Technology (Danvers, MA). All secondary antibodies were obtained from Jackson Immuno-Research Laboratories (West Grove, PA). KN-93 (CalBiochem, La Jolla, CA) is a calmodulin antagonist, specific for a subset of CaM kinases including CaMK-I, CaMK-II and CaMK-IV [ Sumi et al., 1991 ; Hidaka and Ishikawa, 1992 ]. However, CaMK-I and CaMK-IV were undetectable in NIH/3T3 cells as determined using immunological methods (data not shown). KN-93 inhibits CaMK-II by 50% at ∼2 μM and by 90% at 10 μM in these cells [ Tombes et al., 1995 ]. Myristoylated autoinhibitory peptide (KKALRRQEAVDAL) is based on the CaMK-II autoinhibitory domain [ Laabich and Cooper, 2000 ]. Myr-AIP inhibits purified CaMK-II by 90% at 20 μM (data not shown) and was obtained from Biomol (Plymouth Meeting, PA). According to the manufacturer and consistent with our findings, this peptide has an effective treatment limit of 6-8 h.

Supplementary Material Movie 1 Movie 2 Movie 3 Movie 4