Binding proteins to the Src homology 3 (SH3) domains of Nck were screened by the use of glutathione S-transferase fusion proteins. Two proteins of 140 and 125 kDa were detected, both of which associated preferentially with the first SH3 domain of Nck. The 125-kDa protein, designated as Nap1 for Nck-associated protein 1, was purified and the corresponding rat cDNA was isolated. The predicted amino acid sequence revealed that p125Nap1 does not contain any known functional motif but shows sequence homology to Hem family genes. Using specific antibodies, p125Nap1 was shown to associate with Nck both in vitro and in intact cells. Further characterization of p125Nap1 may clarify the protein-protein interaction in the downstream signaling of Nck (Kitamura, 1996).

Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5'-[gamma-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE are indistinguishable, and the 125 kDa protein is depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 react with the 125 kDa protein. Nap1 is co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, can be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase (Kitamura, 1997).

Expression profiles of thousands of genes (cDNAs) were analyzed in sporadic Alzheimer disease (AD)-affected brains in comparison with normal subjects by using the high-density cDNA filter method and differential display analysis. Among 31 differentially expressed genes, one gene was found to be markedly depressed in AD-affected brains. A full-length (or nearly full-length) cDNA of the gene was isolated and sequenced. The cDNA turned out to be an ortholog of rat Nap1. The gene was thus designated human Nap1 (HGMW-approved symbol NCKAP1) and was mapped to human chromosome 2q32 by fluorescence in situ hybridization. Northern blotting and in situ hybridization studies show that in brain, the gene is predominantly expressed in neuronal cells. Antisense oligo DNA of human Nap1 transcripts was found to induce apoptosis of neuronal cells. Based on these results, the possible role of human Nap1 in AD is discussed (Suzuki, 2000).

Expression of apoptosis-related gene human Nap1 (HGMW-approved symbol NCKAP1) is strongly down-regulated in sporadic Alzheimer's disease (AD). Human Nap1 is an ortholog of rat Nap1 which binds to the adaptor molecule Nck in signal transduction. In order to further elucidate the function of human Nap1, yeast two-hybrid screening was performed. A protein designated hNap1BP (human Nap1 binding protein) was discovered that is a member of the tyrosine kinase-binding protein family. In addition, hNap1BP binds to the SH3 domain of c-Abl and Nck. hNap1BP is expressed ubiquitously in various tissues like human Nap1, and intriguingly these genes are co-expressed in hippocampus and cerebral cortex in mouse brain where AD pathological features are strongly evident. Further functional analysis of hNap1BP may clarify its contribution to AD pathology (Yamamoto, 2001).

During body morphogenesis precisely coordinated cell movements and cell shape changes organize the newly differentiated cells of an embryo into functional tissues. Two genes, gex-2 and gex-3, are described whose activities are necessary for initial steps of body morphogenesis in Caenorhabditis elegans. In the absence of gex-2 and gex-3 activities, cells differentiate properly but fail to become organized. The external hypodermal cells fail to spread over and enclose the embryo and instead cluster on the dorsal side. Postembryonically gex-3 activity is required for egg laying and for proper morphogenesis of the gonad. GEX-2 and GEX-3 proteins colocalize to cell boundaries and appear to directly interact. GEX-2 and GEX-3 are highly conserved, with vertebrate homologs implicated in binding the small GTPase Rac and a GEX-3 Drosophila homolog, HEM2/NAP1/KETTE, that interacts genetically with Rac pathway mutants. These findings suggest that GEX-2 and GEX-3 may function at cell boundaries to regulate cell migrations and cell shape changes required for proper morphogenesis and development (Soto, 2002).

To screen for important molecules that interact with a gene of interest in Caenorhabditis elegans (C. elegans), a novel functional screening system was established using the yeast two-hybrid system with the RNA interference technique. This screening system makes it possible to identify the molecular machinery involved in the function of a gene of interest starting with the cDNA of this gene. As a model case, the molecular machinery involved in the function of GEX-3, an essential factor of tissue morphogenesis, was examined. Many interacting molecules were identified by yeast two-hybrid screening and some functional interactions were detected using this novel functional screening system. One such identified interactor is profilin, which not only binds to GEX-3 but also enhances actin nucleation (Tsuboi, 2002).

Nap, a mammalian Kette homolog, exists in a complex with Scar/Wave

Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein) -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. This study reports a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 [p53-inducible messenger RNA with a relative molecular mass (Mr) of 140,000], Nap125 (NCK-associated protein with an Mr of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. It is therefore proposed that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation (Eden, 2002).

Members of the Rho family of small GTPases, such as Cdc42 and Rac1, and of the Src homology (SH) domain-containing SH2-SH3 adapter protein family, such as NCK, link extracellular signals and actin nucleation through pathways that include the WASP family of proteins and the actin nucleation machinery—the Arp2/3 complex. All WASP family members contain a conserved verprolin-homology, cofilin-homology, acidic (VCA) domain that directly binds and activates the Arp2/3 complex. The Arp2/3 complex, in turn, catalyses the nucleation of actin filaments. To prevent undesirable spontaneous actin nucleation in the absence of input signals, the activity of the WASP proteins is tightly regulated. For example, N-WASP is found predominantly in an autoinhibited conformation in which the carboxy-terminal VCA domain is occluded through interaction with the amino terminus of the protein. When Cdc42 binds to the Cdc42/Rac1 interactive binding (CRIB) domain of N-WASP or when NCK binds to the polyproline region of N-WASP, this autoinhibition is relieved and the VCA domain is unmasked. Phosphatidylinositol(4,5)bisphosphate (PIP2) can further activate N-WASP in cooperation with NCK or Cdc42 by binding to a basic region of N-WASP (Eden, 2002).

The WAVE proteins (WAVE1, WAVE2 and WAVE3 in mammals and orthologues in Drosophila and Dictyostelium are similar in structure to N-WASP. They all have a C-terminal VCA domain, a polyproline region and a basic region. Unlike N-WASP, WAVE proteins do not contain a CRIB domain, and direct binding of WAVE1 to Rac1 has not been detected. But much evidence suggests that WAVE1 functions downstream of Rac1. WAVE1 is translocated from the cytoplasm to membrane ruffles induced by Rac1, and dominant-negative WAVE1 abolishes the formation of Rac1-dependent lamellipodia and Rac1-dependent neurite extensions. The mechanism of regulation of WAVE1 is likely to be fundamentally different from that of N-WASP: whereas N-WASP is autoinhibited, recombinant WAVE1 is constitutively active in stimulating the actin nucleation activity of Arp2/3. Therefore, WAVE1 activity is either inhibited in trans by other cellular regulators or regulated by post-translational modifications (Eden, 2002).

To identify potential trans-inhibitory proteins for WAVE1 activation, proteins were sought that interact with WAVE1. All of the detectable WAVE1 protein in soluble bovine brain extracts elutes in size-exclusion chromatography as a complex with an Mr of 500K. This complex was purified to about 90% purity as judged by SDS polyacrylamide gel electrophoresis and Coomassie blue staining. Three proteins co-fractionated and were subsequently co-immunoprecipitated with WAVE1 (Eden, 2002).

The proteins were identified unambiguously by mass spectrometry as the bovine orthologues of the following human proteins: WAVE1; PIR121; Nap125, and HSPC300, which encodes a protein with an Mr of 9K. To test whether WAVE1 activity is inhibited in trans by the other proteins in the complex, the ability of the complex to activate Arp2/3 was tested. Whereas recombinant WAVE1 activates the Arp2/3 complex, the native WAVE1 complex does not. Rac1 is fully effective in relieving the inhibition of WAVE1 in the complex (Eden, 2002).

Although WAVE1 had been implicated as the downstream target of Rac1, no regulatory linkage had been found previously. Consequently, the important Rac1-dependent pathway for actin nucleation has not been described. Similarly, although the association of NCK and Rac1 with NAP125 and PIR121 has been observed in several screens, their role as regulators of actin nucleation has not been shown. These results indicate that WAVE1, like N-WASP and WASP, mediates signals from NCK and the Rho GTPases. The activation mechanisms of WAVE1 and N-WASP are very different: N-WASP is autoinhibited, whereas WAVE1 is trans-inhibited. The action of Rac1 and NCK is to disassemble the trans-inhibited WAVE1 complex, which releases the active WAVE1 protein in association with HSPC300. Consistent with this model, Rac1 and WAVE1 do not colocalize in the lamellipodium: WAVE1 is localized at the extreme edge of the lamellipodium, whereas Rac1 is distributed diffusely over the lamellipodium (Eden, 2002).

Although the predominant regulation of WAVE1 activity described here is relief of trans-inhibition, an additional positive regulation by proteins that bind WAVE1 directly in an activator-independent manner is also possible. For example, IRSp53 has been reported to bind WAVE2 directly and enhance activation of Arp2/3 by recombinant WAVE2. Preliminary data show that HSPC300, which remains associated with WAVE1 after activation, may also have a stimulating function on actin polymerization. The activation and dissociation of the WAVE1 complex process releases a subcomplex of NAP125 and PIR121, and this subcomplex may be free to interact with other cellular components. In this way, a Rac1 or NCK signal might potentially coordinate several cellular processes -- similar to pathways that are activated by the alpha- and the gamma-subunits in heterotrimeric G-protein signalling (Eden, 2002).

Axis specification and morphogenesis in the mouse embryo require Nap1, a regulator of WAVE-mediated actin branching

Dynamic cell movements and rearrangements are essential for the generation of the mammalian body plan, although relatively little is known about the genes that coordinate cell movement and cell fate. WAVE complexes are regulators of the actin cytoskeleton that couple extracellular signals to polarized cell movement. Mouse embryos that lack Nap1, a regulatory component of the WAVE complex, arrest at midgestation and have defects in morphogenesis of all three embryonic germ layers. WAVE protein is not detectable in Nap1 mutants, and other components of the WAVE complex fail to localize to the surface of Nap1 mutant cells; thus loss of Nap1 appears to inactivate the WAVE complex in vivo. Nap1 mutants show specific morphogenetic defects: they fail to close the neural tube, fail to form a single heart tube (cardia bifida), and show delayed migration of endoderm and mesoderm. Other morphogenetic processes appear to proceed normally in the absence of Nap1/WAVE activity: the notochord, the layers of the heart, and the epithelial-to-mesenchymal transition (EMT) at gastrulation appear normal. A striking phenotype seen in approximately one quarter of Nap1 mutants is the duplication of the anteroposterior body axis. The axis duplications arise because Nap1 is required for the normal polarization and migration of cells of the Anterior Visceral Endoderm (AVE), an early extraembryonic organizer tissue. Thus, the Nap1 mutant phenotypes define the crucial roles of Nap1/WAVE-mediated actin regulation in tissue organization and establishment of the body plan of the mammalian embryo (Rakeman, 2006; full text of article).

Nap1 regulates Dictyostelium cell motility and adhesion through SCAR-dependent and -independent pathways

SCAR (also known as WAVE) is a key regulator of actin dynamics. Activation of SCAR enhances the nucleation of new actin filaments through the Arp2/3 complex, causing a localized increase in the rate of actin polymerization. In vivo, SCAR is held in a large regulatory complex, which includes PIR121 and Nap1 (Nck-associated protein 1) proteins, whose precise role is unclear. It was initially thought to hold SCAR inactive until needed, but recent data suggest that it is essential for SCAR function. This study shows that disruption of the gene that encodes Nap1 (napA) causes loss of SCAR function. Cells lacking Nap1 are small and rounded, with diminished actin polymerization and small pseudopods. Furthermore, several aspects of the napA phenotype are more severe than those evoked by the absence of SCAR alone. In particular, napA mutants have defects in cell-substrate adhesion and multicellular development. Despite these defects, napA- cells move and chemotax surprisingly effectively. These results show that the members of the complex have unexpectedly diverse biological roles (Ibarra, 2006)

An actin-based wave generator organizes cell motility

Although many of the regulators of actin assembly are known, how these components act together to organize cell shape and movement is not understood. To address this question, the spatial dynamics were analyzed of a key actin regulator-the Scar/WAVE complex-which plays an important role in regulating cell shape in both metazoans and plants. The Hem-1/Nap1 component of the Scar/WAVE complex localizes to propagating waves that appear to organize the leading edge of a motile immune cell, the human neutrophil. Actin is both an output and input to the Scar/WAVE complex: the complex stimulates actin assembly, and actin polymer is also required to remove the complex from the membrane. These reciprocal interactions appear to generate propagated waves of actin nucleation that exhibit many of the properties of morphogenesis in motile cells, such as the ability of cells to flow around barriers and the intricate spatial organization of protrusion at the leading edge. It is proposed that cell motility results from the collective behavior of multiple self-organizing waves (Weiner, 2007; full text of article).

Nap1-regulated neuronal cytoskeletal dynamics is essential for the final differentiation of neurons in cerebral cortex

The cytoskeletal regulators that mediate the change in the neuronal cytoskeletal machinery from one that promotes oriented motility to one that facilitates differentiation at the appropriate locations in the developing neocortex remain unknown. This study found that Nck-associated protein 1 (Nap1), an adaptor protein thought to modulate actin nucleation, is selectively expressed in the developing cortical plate, where neurons terminate their migration and initiate laminar-specific differentiation. Loss of Nap1 function disrupts neuronal differentiation. Premature expression of Nap1 in migrating neurons retards migration and promotes postmigratory differentiation. Nap1 gene mutation in mice leads to neural tube and neuronal differentiation defects. Disruption of Nap1 retards the ability to localize key actin cytoskeletal regulators such as WAVE1 to the protrusive edges where they are needed to elaborate process outgrowth. Thus, Nap1 plays an essential role in facilitating neuronal cytoskeletal changes underlying the postmigratory differentiation of cortical neurons, a critical step in functional wiring of the cortex (Yokota, 2007).

HEM-protein/Kette: Biological Overview | Developmental Biology | Effects of Mutation | References

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