Nck/Dock: an adapter between cell surface receptors and the actin cytoskeleton

119

Rho-like GTPases control a wide range of cellular functions such as integrin-and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the Cterminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.Rho-like GTPases drive temporally and spatially coordinated actin polymerization to control cell motility, cadherin-based cell-cell adhesion, and cytokinesis (1-4). To relay their signals, Rho-like GTPases interact with a wide range of effector proteins. These interactions are generally thought to occur at the plasma membrane to which GTPases are translocated prior to activation by guanine nucleotide exchange factors. This membrane association is dependent on lipid modifications of specific cysteine residues in the extreme C terminus (i.e. prenylation), as well as on the C-terminal polybasic region (5).Rho-like GTPases share a high level of homology, despite the fact that their effects on cellular morphology or function can be very distinct. This has been attributed to sequence diversity in the effector domain (residues 26 -45) and the so-called insert region (amino acids 124 -135). However, the most divergent region between otherwise very homologous GTPases (e.g. Rac 1066 CX, and Rac2) is the C-terminal polybasic region. This domain was shown to be required for activation of the neutrophil NADPH oxidase (6) and was claimed for Rac1 to drive activation of PAK 1 by some (7) but not by others (6). Most of the studies that have addressed the role of this domain relied on deletion or mutation strategies, testing the role of this region in the context of, for instance, a constitutively active variant of the GTPase.Tao et al. (5) showed recently that deleting the C-terminal polybasic region affects the efficiency of prenylation of Rac2. Moreover, del Pozo et al. (8) have shown that mutation of the crucial cysteine residue to prevent Rac prenylation blocked activation of PAK. This means that mutating or deleting the C-terminal domain may affect downstream signaling in an indirect manner, complicating the interpret...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The C-terminal Domain of Rac1 Contains Two Motifs That Control Targeting and Signaling Specificity

Hennik

Klooster

Halstead

et al. 2003

119

“…Indeed, under resting conditions, PAK1 to -3 interact with the Nck adapter that upon activation links tyrosine kinase receptors and brings PAK to the membrane (for a review, see Ref. 22). However, PAKs also form stable complexes with the guanine exchange factors for Rac and Cdc42 of the PIX/Cool family, and these interactions are not only closely involved in targeting PAK to the membrane but also in regulating its kinase activity (23)(24)(25).…”

mentioning

confidence: 99%

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Rousseau¹,

Goupille²,

Morin³

et al. 2003

p21-activated kinases (PAK) are involved in the control of cytoskeleton dynamics and cell cycle progression. Here we report the characterization of a new mammalian PAK3 mRNA that contains a 45-bp alternatively spliced exon. This exon encodes for 15 amino acids that are inserted in the regulatory domain, inside the autoinhibitory domain but outside the Cdc42 and Rac interactive binding domain. The transcript of the 68-kDa new isoform named PAK3b is expressed in various areas of the adult mouse brain. In contrast to PAK3 without the exon b (PAK3a), whose basal kinase activity is weak in resting cells, PAK3b displays a high kinase activity in starved cells that is not further stimulated by active GTPases. Indeed, we demonstrate that the autoinhibitory domain of PAK3b no longer inhibits the kinase activity of PAK3. Moreover, we show that the 15-amino acid insertion within the autoinhibitory domain impedes the ability of PAK3b to bind to the GTPases Rac and Cdc42 and changes its specificity toward the GTPases. Altogether, our results show that the new PAK3b isoform has unique properties and would signal differently from PAK3a in neurons.

“…The ETA Receptor Induces the Inhibition of Cell Migration through Nck1 but Not through Grb2 or CrkII-A growing number of studies have suggested that adaptor proteins play an important role in mediating the signaling pathway from receptor tyrosine kinases to the Rho family-dependent regulation of the actin cytoskeleton (25,26). Cell migration is a complex cellular process that is regulated by a number of regulatory proteins and is driven by cytoskeletal reorganization.…”

Section: Resultsmentioning

confidence: 99%

The Adaptor Protein Nck1 Mediates Endothelin A Receptor-regulated Cell Migration through the Cdc42-dependent c-Jun N-terminal Kinase Pathway

Miyamoto

Yamauchi

Mizuno

et al. 2004

Cell migration plays key roles in physiological and pathological phenomena, such as development and oncogenesis. The adaptor proteins Grb2, CrkII, and Nck1 are composed of only a single Src homology 2 domain and some Src homology 3 domains, giving specificity to each signal transduction pathway. However, little is known about the relationships between their adaptor proteins and cell migration, which are regulated by the G protein-coupled receptor. Here we showed that Nck1, but not Grb2 or CrkII, mediated the inhibition of cell migration induced by the endothelin-1 and endothelin A receptor. The small interference RNA and dominant negative mutants of Nck1 diminished the endothelin-1-induced inhibition of cell migration. Although overexpression of wild-type Nck1 was detected in the cytosol and did not affect cell migration, expression of the myristoylation signal sequence-conjugated Nck1 was detected in the membrane and induced activation of Cdc42 and c-Jun N-terminal kinase, inhibiting cell migration. Taken together, these results suggest that the endothelin A receptor transduces the signal of inhibition of cell migration through Cdc42-dependent c-Jun N-terminal kinase activation by using Nck1.