Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity
Graphical abstract
Introduction
The molecular switcher Ras is regulated by GDP/GTP exchange factors (GEFs) and Ras GTPase activating proteins (RasGAPs) and is implicated in cell proliferation, differentiation and apoptosis control [1].
The p120 RasGAP, negative regulator of Ras, has COOH-terminal catalytic domain and NH2-terminal part consisting of proline-rich sequence, SH2 domains, SH3 domain, PH domain and other non-catalytic domains, which regulate association with a number of proteins [2]. RasGAP can act upstream of Ras, as its down-regulator, and downstream of Ras, as its effector. Upon ligand-activation platelet-derived growth factor (PDGF) receptor-β associates with RasGAP [3], down-regulates Ras [4] and attenuates PI3-kinase activation [5]. On the other hand, RasGAP and p190 Rho-GAP constitutive association is required for Ras-independent directional cell movement [6]. RasGAP binds to Aurora kinase domain and inhibits its kinase activity in Ras-independent mode [7]. RasGAP also is involved in caspase-dependent and independent apoptosis regulation [8], [9], angiogenesis [10] and presumably protein synthesis [11].
Regulation of RasGAP catalytic activity is still poorly understood. Herpes simplex virus type 2 protein kinase, ICP10 [12] or c-Src [13] phosphorylates and inhibits RasGAP. The PH domain of RasGAP associates with catalytic domain and attenuates it activity [14]. The p62-Dok or p190-RhoGAP interacts with RasGAP and inhibits it activity [15], [16]. No positive regulators of RasGAP catalytic activity have been reported up to date.
The SH2/SH3 domain-containing adaptor proteins Nck1 and Nck2 mediate membrane receptor signaling toward cytoskeleton rearrangement [17], [18], septin-depended cell cycle arrest and apoptosis [19], as well as integrate endogenous stress signals by inhibition of protein translation [20], [21]. Adaptor protein Nck acts as scaffold protein, however, no effect on other protein catalytic activity have been reported yet.
Here we provide evidence that Nck1 directly associates with RasGAP and increases its catalytic activity toward H-Ras; cell adhesion to the substrate is necessary for the complex formation, as detachment of cells leads to the complex dissociation that correlates with decrease of RasGAP activity and to the increase of Ras-GTP level, suggesting a role for Nck1-RasGAP complex in anoikis regulation.
Section snippets
Cell culture and antibodies
Human hepatocellular carcinoma HepG2 cell line ectopically expressing human PDGF receptor-β, NIH3T3, mouse embryo fibroblasts, and Myo27, rabbit primary myogenic cells were maintained in Dulbecco's modified Eagle's medium (Biochrom) supplemented with 10% fetal bovine serum (Sigma) and antibiotics as previously described [22].
The rabbit polyclonal antibodies (Ab) specific for Nck, GST and RasGAP were raised in our laboratory. Anti-Ras monoclonal antibody (mAb) was obtained from BD Transduction
Adaptor protein Nck1 interacts with RasGAP
Previously we have provided evidence that recombinant protein Nck1 is capable of binding endogenous RasGAP [28]. Here we asked whether the endogenous Nck1 protein can form complex with RasGAP in intact cells and how cell stimulation with the platelet-derived growth factor BB (PDGF) affects complex formation.
Nck1 protein was immunoprecipitated from the PDGF-stimulated or resting HepG2 cells which ectopically express PDGF receptor-β. The immunoprecipitate was subjected to anti-RasGAP or anti-Nck
Discussion
In this study we elucidated the mechanism of Nck1 and RasGAP association and investigated the possible role of Nck1 and RasGAP complex formation in the regulation of H-Ras activity.
It has been shown earlier that Nck can be implicated in the Ras regulation. For example, Nck binds Sos1 and might contribute to the increase of Ras-dependent c-fos expression [33]. Also Nck and RasGAP interact with tyrosine-phosphorylated p62-Dok adaptor protein [34], which negatively regulates Ras activity [15]. Nck
Conclusions
In summary, we elucidated the mechanism of interaction between Nck1 and RasGAP and for the first time demonstrated that RasGAP activity can be increased by association with adaptor protein Nck1. Albeit complex formation is constitutive and directed via SH3 domains of Nck1 and proline-rich sequence of RasGAP, it dissipates upon cell detachment from the substrate. Complex dissipation is consistent with the decrease of RasGAP catalytic activity and with the increase in Ras activity, suggesting the
Acknowledgments
We would like to thank V. Gurskienė, A. Marandykina and V. Tunaitis for excellent technical assistance. We are grateful to W. Li (University of Southern California, USA), L.A. Quilliam (Indiana University School of Medicine, USA) and T. Kataoka (Kobe University Graduate School, USA) for the generous provision of plasmids. This work was supported by Vilnius University Institute of Biochemistry funding. Authors report no conflict of interests.
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