Nck Adapters Are Involved in the Formation of Dorsal Ruffles, Cell Migration, and Rho Signaling Downstream of the Platelet-derived Growth Factor β Receptor

Aino Ruusala,Tony Pawson,Pontus Aspenström,Carl-Henrik Heldin

doi:10.1074/jbc.m800913200

Aino Ruusala, Tony Pawson + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m800913200

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Abstract

The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFbeta receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFbeta receptor.

Highlights

platelet-derived growth factor (PDGF) receptor constitute docking sites for SH2 domain-containing proteins [1, 2]
The role of the individual tyrosine residues in PDGF signaling has been extensively studied in cells such as fibroblasts and porcine aortic endothelial (PAE) cells expressing mutant PDGFR␤ in which specific tyrosine residues in the cytoplasmic part of the PDGFR␤ have been replaced with phenylalanine residues [1, 2]
In an effort to study the involvement of the Nck adaptor proteins in PDGF signaling separate from phosphoinositide 3-kinase (PI3K)-mediated signaling, we used mouse embryonic fibroblasts (MEFs) in which the Nck1 and Nck2 genes had been inactivated by gene targeting (KO cells) [19]

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Plasmids—Rabbit polyclonal anti-Arp3A expression was triggered by transfection of WT MEFs with antiserum was a generous gift from L. The cells on the coverslips were fixed in 2% paraformaldehyde in PBS for 20 min at room temperature. For Western blotting analysis, cell lysis was performed essentially as described before [20]. Western blotting was performed with primary mouse or rabbit antibodies followed by horseradish peroxide-conjugated antimouse or anti-rabbit secondary antibodies (GE Healthcare). After washing the wells once with PBS, the attached cells were fixed for 10 min in 96% ethanol at room-temperature, stained with crystal violet for 30 min, washed, and lysed in 1% Triton X-100 for 20 min on a shaker. Activity assays for activated, GTPbound, Cdc, Rac, and RhoA were performed essentially as described before [23]

RESULTS

Cells Lacking Nck Adapters Have a Reduced Ability to Form Focal

Defective in Cells Lacking Nck

We next stimulated WT cells with

DISCUSSION