Abstract
Extracellular signal-regulated kinase 1/2 (ERK1/2) is activated by various extracellular stimuli including growth factors and cytokines and plays a pivotal role in regulating cell proliferation and differentiation by phosphorylating nuclear transcription factors. Recently, it was reported that activated ERK1/2 also concentrates at adhesion sites and regulates cell spreading and migration. Vinexin is a focal adhesion protein regulating both cell spreading and growth factor signaling. We show here that vinexin was directly phosphorylated by ERK1/2 upon stimulation with growth factors. ERK1/2 phosphorylated the linker region of vinexin between the second and third SH3 domains. Site-directed mutagenesis revealed that ERK2 mainly phosphorylated the serine 189 residue of vinexin beta. Furthermore, vinexin beta interacted with ERK1/2 both in vitro and in vivo. Vinexin interacted with the active but not inactive form of ERK1/2. A putative DEF (docking for ERK FXFP) domain located in the linker region of vinexin was required for the interaction with ERK1/2 and efficient phosphorylation of vinexin beta by ERK2. Finally, we showed that cell adhesion to fibronectin also induced the association of vinexin beta with ERK2 and the phosphorylation of vinexin beta. Furthermore, vinexin and ERK were co-localized to the periphery of cells during cell spreading on fibronectin. Together, these results suggest that vinexin is a novel substrate of ERK2 and may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.
Highlights
Mitogen-activated protein kinase (MAPK)1 consists of four subfamilies, extracellular signal-regulated kinase 1/2 (ERK1/ 2), c-Jun N-terminal kinase/stress-activated kinase, p38MAPK, and ERK5
We previously showed that expression of vinexin ␣ affected the actin cytoskeletal organization in NIH3T3 cells, and that expression of vinexin  promoted the spreading of C2C12 cells [18]
We showed that vinexin was phosphorylated downstream of the MEK-ERK cascade upon stimulation with EGF, and that vinexin  was phosphorylated directly by ERK2 in vitro
Summary
Cell Culture and Transfection—NIH3T3 cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum and COS7 cells, A431 cells, HT1080 cells, and HeLa cells were cultured with DMEM supplemented with 10% fetal bovine serum. They were stimulated with serum (10%), EGF (100 ng/ml), PDGF (50 ng/ml), TPA (50 g/ml), LPA (0.5 mg/ml), or insulin (100 M) for 5 min and lysed with lysis buffer (1% Triton X-100, 0.02 mg/ml aprotinin, 0.1 mg/ml p-amidinophenyl methanesulfonyl fluoride hydrochloride, 5 g/ml leupeptin, 5 mM benzamidine, 1 g/ml pepstatin A, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 40 mM -glycerophosphate, and 20 nM calyculin A in PBS). In Vitro Binding Assay—GST or GST-fused proteins (5 g) were incubated with active or inactive ERK2 (25 pmol) in 1% Triton X-100/ PBS at 4 °C for 60 min. The cells were blocked with 10% goat serum/TBS-T (TBS with 0.1% Triton X-100) for 1 h, incubated with anti-vinexin antibody and anti-panERK antibody, or anti-phospho-ERK1/2 antibody at 4 °C overnight. Fluorescence images were taken with LSM 5 PASCAL confocal microscopy system (Carl Zeiss Co., Ltd)
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