Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

N-WASP

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_605

Synonyms

Historical Background

Wiskott–Aldrich syndrome (WAS), a pediatric disorder, was first described in 1937 by Alfred Wiskott as a “hereditary thrombopathy” in males, presenting with thrombocytopenia, eczema, bloody diarrhea, episodes of fever, and recurrent bacterial infections. Robert A. Aldrich would later demonstrate an X-linked mode of inheritance of this disease. Other features of WAS were later recognized, including immunodeficiency involving both humoral and cellular immunity, high rate of autoimmunity and malignancies, abnormal apoptosis, and defective cell motility. The mutated gene giving rise to this disease was identified in 1994 by positional cloning and referred to as WAS, and mutations of the WAS protein (WASP) were demonstrated not only in patients with WAS, but also in those with X-linked thrombocytopenia (XLT), a disease showing milder clinical phenotype with a more favorable prognosis (Notarangelo et al. 2008). Northern blot analysis indicated that WASP mRNA is expressed exclusively in hematopoietic cells. Two years later, a novel protein with ∼50% amino acid identity to the WAS gene product was reported as a binding partner for the Grb2/Ash adapter protein. In contrast to WASP, this protein was expressed ubiquitously, but strongest expression was observed in neuronal cells and was thus named Neural-WASP ( N-WASP) (Miki and Takenawa 2003).

 N-WASP and WASP are actin-nucleating promoting factors (NPF) and are the founding members of the WASP-family of NPFs that contain tandem V (Verprolin homology, also known as WH2; WASP homology 2), C (Central or Connecting), and A (Acidic) regions, referred to as the VCA domain. Other members of this family are the WAVE/Scar, WASH, and WHAMM/JMY subfamilies. Evolutionary analyses indicate that these VCA domain-containing proteins are widely expressed among eukaryotes and evolutionarily ancient (Veltman and Insall 2010).

Structure of N-WASP

 N-WASP and WASP have a conserved domain organization that allows interaction with multiple distinct binding proteins (Table 1). They have a WASP homology 1 (WH1; also known as EVH1) domain that binds primarily WASP interacting protein (WIP)-family members, a basic sequence that binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), a GTPase-binding domain (GBD), containing a Cdc42 and Rac interactive binding (CRIB) motif that binds Cdc42-GTP, and a proline-rich region that binds SH3 proteins. The C-terminus of both proteins contains a VCA domain that binds actin via the V region and the Arp2/3 complex via the CA region but  N-WASP has an additional V region (Takenawa and Suetsugu 2007) (Fig. 1). In vitro data using the purified VVCA domain of  N-WASP showed that this domain alone was sufficient for actin polymerization and its activity was higher than the isolated VCA domain of WASP (Zalevsky et al. 2001).
N-WASP, Table 1

 N-WASP/WASP binding proteins

Binding protein

Binding domain

N-WASP/WASP binding region

Effect on (N)WASP/function

WIP, CR16, WICH/WIRE

Proline-rich

WH1

Stabilization, targeting, inhibition of activity

CIB

 

WH1

Coupling to integrin αIIbβ3

Cdc42/Chp

 

CRIB

Activation

TC10/RhoT

 

CRIB

Activation

mDab1

PTB

CRIB

 

PtdIns(4,5)P2

 

Basic

Localization, synergistic activation with Cdc42 and Nck

Hsp90

 

Basic

Stabilization

FBP11

WW

Proline-rich

 

Src-family kinases (Src, Hck, Lck, Lyn, Fyn, Fgr)

SH3 (SH2?)

Proline-rich

Phosphorylation, activation, protein degradation

Grb2

SH3

Proline-rich

Activation, localization

Nck

SH3

Proline-rich

Activation, localization

Crk adaptors (CrkII, CrkL)

SH3

Proline-rich

Activation, localization

Vinexin beta

SH3

Proline-rich

 

Cortactin, HS1

SH3

Proline-rich

Targeting, activation, invadopodium/podosome formation

Abi1

SH3

Proline-rich

Activation, endocytosis

Endophilin

SH3

Proline-rich

Activation, endocytosis

Amphiphysin

SH3

Proline-rich

Endocytosis

Tuba

SH3

Proline-rich

Activation via Cdc42

CIP4, Toca-1, FBP17

SH3

Proline-rich

Cdc42-mediated activation

Tec family kinases (Tec, Btk, Itk)

SH3

Proline-rich

Phosphorylation, activation

WISH/DIP

SH3

Proline-rich

Activation

PSTPIP1

SH3

Proline-rich

Activation, localization

Syndapin

SH3

Proline-rich

Activation, endocytosis

Nostrin

SH3

Proline-rich

Endocytosis

srGAP

SH3

Proline-rich

 

Profilin

SH3

Proline-rich

Actin polymerization

IRSp53

SH3

Proline-rich

Activation, localization

PTP-PEST

 

Proline-rich

Dephosphorylation, inactivation

Sorting nexin (SNX9, SNX18)

SH3

Proline-rich

Activation, endocytosis

Arg

SH3

Proline-rich

Phosphorylation, activation

Intersectin-1, -2

SH3

Proline-rich

Localization, activation

Abp1

SH3

Proline-rich

Activation, endocytosis

VASP

 

Proline-rich

Localization, actin polymerization

Casein kinase

?

?

VCA phosphorylation

Arp2/3

 

CA

Actin polymerization

G-actin

 

V

Actin polymerization

F-actin

 

Basic

Branching

Merlin, ERM (ezrin/radixin/moesin)

FERM domain

WH1

Inhibition of actin polymerization

CD44

  

Stabilization, localization

IQGAP1

 

Basic-CRIB

Activation

? unknown

N-WASP, Fig. 1

Domain organization and molecular interactions of  N-WASP and WASP. N-WASP and WASP have similar overall domain organization, with an additional V region present in  N-WASP. The WH1 domain interacts with WIP and its related proteins. The basic domain mediates interactions with PtdIns(4,5)P2 on membranes. The CRIB motif inside the GBD region binds to Cdc42-GTP and is instrumental in  N-WASP activation. The polyproline stretch is a site for docking of SH3 domains, while distinct regions mediate binding to profilin. Finally, the VCA domain binds G-actin and the Arp2/3 complex and nucleates actin polymerization

Regulation of N-WASP

Autoinhibition and Activation by Cdc42

 N-WASP is regulated by diverse signals including Rho family GTPases, phospholipids, kinases, many SH3 domain-containing proteins, and both bacterial and viral pathogen proteins (Takenawa and Suetsugu 2007) (Table 1). In the resting state,  N-WASP and WASP are autoinhibited through intramolecular interactions between the C-terminal VCA region and a hydrophobic pocket in the GBD but other regions, such as from the WH1 domain, may also participate in autoinhibition. In addition, it is generally considered that favorable electrostatic interactions between the basic region and the A region further stabilize the GBD–VCA interface. The Rho family GTPase Cdc42 was the first protein shown to bind  N-WASP/WASP and is an important regulator of both proteins. The active, GTP bound form of Cdc42 disrupts the hydrophobic core of the GBD and releases the VCA domain, which can now bind G actin and the Arp2/3 complex. The Arp2/3 complex, which is composed of seven polypeptides, nucleates actin polymerization and contributes to branched filamentous actin (Takenawa and Suetsugu 2007). Another Rho family GTPase, Rac1, can activate  N-WASP in vitro, but is probably not involved in WASP activation (Tomasevic et al. 2007). Evidence also suggests that Cdc42 may require an additional intermediate for the activation of  N-WASP, such as F-BAR proteins of the CIP4 subfamily, which include Toca-1, CIP4, and FBP17. These proteins bind directly to Cdc42 and also contain a SH3 domain that binds to the polyproline region of  N-WASP, and is required for  N-WASP functions, such as endocytosis.

Regulation by Phosphorylation

Although it is generally accepted that the binding of Cdc42-GTP to WASP is important for allosteric release from autoinhibition, other signaling events can also modulate  N-WASP function. One such event is phosphorylation of a conserved tyrosine residue (Y256 in human  N-WASP and Y291 in human WASP) by non-receptor tyrosine kinases, e.g.,  Src, and is required for several cellular functions of  N-WASP, such as neurite extension. Remarkably, this residue is located in the C-terminus of the GBD, and its phosphorylation is thought to alter the charge and therefore stability of the autoinhibited form of both proteins shifting the proteins toward the open, active conformation (Thrasher and Burns 2010). Additionally, tyrosine phosphorylation may prime the molecule for activation by proteins that contain SH2 domains contributing to an additional regulatory input. However, tyrosine phosphorylation may also target both WASP and  N-WASP to various protein degradation pathways, thus resulting in signal termination (Dovas and Cox 2010).

PtdIns(4,5)P2, SH3 Domains, and EspFu

Additional levels of control of  N-WASP activity following allosteric activation also exist, facilitating the integration of multiple signals in the efficient control of  N-WASP activity. These are generally thought of as influencing the activity of  N-WASP by correctly localizing the molecule and/or by influencing its dimerization status (Padrick and Rosen 2010). The latter is an important means of control of  N-WASP-dependent actin polymerization since VCA domain dimers bind the Arp2/3 complex with higher affinity than monomers, thus contributing to enhanced actin polymerization. PtdIns(4,5)P2 is an important regulator of actin organization mediated by both  N-WASP and WASP. PtdIns(4,5)P2 synergizes with Cdc42 in the activation of  N-WASP. Increased PtdIns(4,5)P2 density hyperactivates  N-WASP in vitro, and is required for actin comet tail formation in vivo, pointing to both membrane targeting and dimerization as means by which PtdIns(4,5)P2 regulates  N-WASP.

Adaptor proteins that contain multiple SH3 domains participate in an additional level of control of  N-WASP activity via dimerization. One prominent example is Nck1, which contains five SH3 domains and is able to hyperactivate  N-WASP in vitro, while dimeric BAR domain proteins that contain SH3 domains may also act by inducing  N-WASP dimers. Importantly, the enhanced activity of  N-WASP upon dimerization is exploited by enterohemorrhagic Escherichia coli, to induce its spread. The bacterial protein EspFu, which is injected into the host cell cytoplasm, contains multiple repeats of a segment that can bind the GBD of  N-WASP. Therefore, EspFu activates  N-WASP allosterically by competing for GBD binding with the VCA domain while at the same time clustering multiple  N-WASP molecules to stimulate Arp2/3-dependent actin polymerization and pedestal formation. Therefore, these data suggest that the proline-rich region may receive activation signals as well as localization signals from proteins containing SH3 domains, and that similar mechanisms are exploited by pathogens to induce their spread.

WIP: A Major Binding Partner

WIP interacts with the WH1 domain of  N-WASP and WASP and performs critical regulatory functions (Ramesh and Geha 2009). Primarily, it is required for the stability of WASP protein levels in vivo. This is highlighted by the fact that the most frequent WAS mutations are clustered at the WH1 domain and affect amino acids important for the interaction with WIP. WIP also targets WASP to sites of activity. For example, WIP targets WASP to sites of T cell receptor clustering and also recruits  N-WASP to vaccinia virus particles. However, WIP also inhibits the ability of  N-WASP to stimulate actin polymerization, at least in vitro. It may be possible that WIP does not dissociate from  N-WASP but may undergo conformational changes that relieve inhibitory interactions and allow  N-WASP activation in vivo (Ramesh and Geha 2009).

The Role of N-WASP

 N-WASP exerts its functions primarily via the regulation of Arp2/3-mediated actin polymerization. As such, it participates in a plethora of actin-dependent functions including endocytosis, phagocytosis, invadopodium assembly, neurite extension, vesicular transport, pathogen infection, and dorsal ruffle formation (Fig. 2).  N-WASP is also found in the nucleus where it participates in gene transcription through association with a nuclear complex that contains RNA polymerase II. This role of  N-WASP also relies on its ability to polymerize nuclear actin (Wu et al. 2006). Similar nuclear functions have been described for WASP, where it is important in the transcription of key genes required for the differentiation of CD4+ TH1 cells (Taylor et al. 2010).  N-WASP also regulates sarcomeric actin assembly in skeletal muscle, though independently of the Arp2/3 complex (Takano et al. 2010).
N-WASP, Fig. 2

Cellular functions of  N-WASP .  N-WASP plays important functions inside cells. It mediates endocytosis and vesicle motility; participates in plasma membrane extensions, such as in the outgrowth of neurites, during phagocytosis, dorsal ruffle formation and invadopodium formation, and regulates gene expression by shuttling in and out of the nucleus

 N-WASP has not been associated with human disease and appears to be essential in mammals as  N-WASP deficiency in mice results in embryonic death at day E12. Tissue-specific ablation of  N-WASP, however, has revealed roles for  N-WASP in T cell development (Cotta-de-Almeida et al. 2007), hair follicle cycling (Lefever et al. 2010), and myelin sheath formation by Schwann cells (Novak et al. 2011). The importance of tight regulation of  N-WASP activity is suggested by the hematopoetic-restricted WASP in which activating mutations have been shown to induce a separate syndrome, X-linked neutropenia (XLN). Therefore, both inactivating and activating mutations in WASP contribute to human disease.

Common and Distinct Functions of N-WASP and WASP

Although there is a high degree of homology in the functional domains of  N-WASP and WASP, they may not be able to completely substitute for one another. WASP and  N-WASP may serve both redundant and nonredundant functions depending on the cellular context. For example, only  N-WASP, and not WASP, can support Shigella motility in cells (Snapper et al. 2001). Also, while WASP is expressed exclusively in hematopoietic cells,  N-WASP is also expressed in these cells, albeit at low levels (Isaac et al. 2010). Recently, unique functions for  N-WASP were demonstrated in macrophages. A striking feature of WASP-deficient macrophages is the lack of podosomes. Podosomes mediate adhesion to the extracellular matrix and perform matrix degradation. WASP localizes to the F-actin-rich core along with other actin-regulatory proteins, such as cortactin/HS1 and Arp2/3. Interestingly, certain aggressive cancer cells and  Src-transformed cells possess podosome-like structures called invadopodia that appear to be directly responsible for extracellular matrix degradation. Invadopodia have similar organization and actin regulatory machinery localization compared to podosomes. However, invadopodia are regulated by  N-WASP given the absence of WASP expression in these cells. When  N-WASP was reduced in macrophage cells, podosomes still formed, but they were unable to perform matrix degradation (Nusblat et al. 2011). This defect was rescued by re-expression of  N-WASP, but not by overexpression of WASP. Additionally, reducing  N-WASP levels mistargets the matrix-degrading enzyme MT1-MMP and it no longer localizes to podosomes. Additionally,  N-WASP only co-localizes with MT1-MMP positive vesicles at podosomes, suggesting that  N-WASP may play a role on the targeting or fusion of MMP-containing vesicles to podosomes in macrophage cells (Nusblat et al. 2011). A unique role for  N-WASP was also found for phagocytosis by macrophages and a study indicated that  N-WASP may play a role in membrane delivery to the growing phagocytic cup while WASP may be required for the actin polymerization during phagocytosis (Park and Cox 2009).  N-WASP and WASP have both overlapping and unique functions when expressed in the same cell.

Summary

Both  N-WASP and WASP proteins play critical roles in rapid reorganization of actin filaments induced in response to diverse extracellular stimuli. Although they have high homology in their functional domains,  N-WASP and WASP also have different requirements for activation and participate in distinct cellular processes. Studies in leukocytes, which express both proteins, may reveal unique functions and distinct aspects of regulation and can contribute to a more profound understanding of their cellular activities and further delineate the immunological abnormalities in WAS. Identification of different binding partners between  N-WASP and WASP will provide important answers to the intriguing questions about the differential activation and unique roles of these proteins. Tissue-specific ablation of  N-WASP will also help reveal its functions in vivo. Novel findings on the nuclear or Arp2/3-independent functions of  N-WASP suggest that many aspects of this molecule remain unknown and promise exciting new avenues for research.

References

  1. Cotta-de-Almeida V, Westerberg L, Maillard MH, et al. Wiskott Aldrich syndrome protein (WASP) and N-WASP are critical for T cell development. Proc Natl Acad Sci USA. 2007;104(39):15424–9.PubMedPubMedCentralCrossRefGoogle Scholar
  2. Dovas A, Cox D. Regulation of WASp by phosphorylation: activation or other functions? Commun Integr Biol. 2010;3(2):101–5.PubMedPubMedCentralCrossRefGoogle Scholar
  3. Isaac BM, Ishihara D, Nusblat LM, et al. N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis. Exp Cell Res. 2010;316(20):3406–16.PubMedPubMedCentralCrossRefGoogle Scholar
  4. Lefever T, Pedersen E, Basse A, et al. N-WASP is a novel regulator of hair-follicle cycling that controls antiproliferative TGF{beta} pathways. J Cell Sci. 2010;123(Pt 1):128–40.PubMedCrossRefGoogle Scholar
  5. Miki H, Takenawa T. Regulation of actin dynamics by WASP family proteins. J Biochem. 2003;134(3):309–13.PubMedCrossRefGoogle Scholar
  6. Notarangelo LD, Miao CH, Ochs HD. Wiskott-Aldrich syndrome. Curr Opin Hematol. 2008;15(1):30–6.PubMedCrossRefGoogle Scholar
  7. Novak N, Bar V, Sabanay H, et al. N-WASP is required for membrane wrapping and myelination by Schwann cells. J Cell Biol. 2011;192(2):243–50.PubMedPubMedCentralCrossRefGoogle Scholar
  8. Nusblat LM, Dovas A, Cox D. The non-redundant role of N-WASP in podosome-mediated matrix degradation in macrophages. Eur J Cell Biol. 2011;90(2–3):205–12.PubMedCrossRefGoogle Scholar
  9. Padrick SB, Rosen MK. Physical mechanisms of signal integration by WASP family proteins. Annu Rev Biochem. 2010;79:707–35.PubMedPubMedCentralCrossRefGoogle Scholar
  10. Park H, Cox D. Cdc42 Regulates Fc gamma receptor-mediated phagocytosis through the activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP) and neural-WASP. Mol Biol Cell. 2009;20(21):4500–8.PubMedPubMedCentralCrossRefGoogle Scholar
  11. Ramesh N, Geha R. Recent advances in the biology of WASP and WIP. Immunol Res. 2009;44(1–3):99–111.PubMedCrossRefGoogle Scholar
  12. Snapper SB, Takeshima F, Anton I, et al. N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nat Cell Biol. 2001;3(10):897–904.PubMedCrossRefGoogle Scholar
  13. Takano K, Watanabe-Takano H, Suetsugu S, et al. Nebulin and N-WASP cooperate to cause IGF-1-induced sarcomeric actin filament formation. Science. 2010;330(6010):1536–40.PubMedCrossRefGoogle Scholar
  14. Takenawa T, Suetsugu S. The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat Rev Mol Cell Biol. 2007;8(1):37–48.PubMedCrossRefGoogle Scholar
  15. Taylor MD, Sadhukhan S, Kottangada P, et al. Nuclear role of WASp in the pathogenesis of dysregulated TH1 immunity in human Wiskott-Aldrich syndrome. Sci Transl Med. 2010;2(37):37–44.CrossRefGoogle Scholar
  16. Thrasher AJ, Burns SO. WASP: a key immunological multitasker. Nat Rev Immunol. 2010;10(3):182–92.PubMedCrossRefGoogle Scholar
  17. Tomasevic N, Jia Z, Russell A, et al. Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2. Biochemistry. 2007;46(11):3494–502.PubMedCrossRefGoogle Scholar
  18. Veltman DM, Insall RH. WASP family proteins: their evolution and its physiological implications. Mol Biol Cell. 2010;21(16):2880–93.PubMedPubMedCentralCrossRefGoogle Scholar
  19. Wu X, Yoo Y, Okuhama NN, et al. Regulation of RNA-polymerase-II-dependent transcription by N-WASP and its nuclear-binding partners. Nat Cell Biol. 2006;8(7):756–63.PubMedCrossRefGoogle Scholar
  20. Zalevsky J, Lempert L, Kranitz H, et al. Different WASP family proteins stimulate different Arp2/3 complex-dependent actin-nucleating activities. Curr Biol. 2001;11(24):1903–13.PubMedCrossRefGoogle Scholar

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Anatomy and Structural BiologyAlbert Einstein College of Medicine, Jack and Pearl Resnick CampusBronxUSA