The WW module competes with the SH3 domain?

FRONTLINES

TIBS 2 1 - MAY 1996

tryptophan residues (W), which are spaced 20-22 amino acids apart.

The WWmodule competes with the SH3 domain? At the recent workshop on Molecular Repertoires and Methods of Selection*, Riccardo Cortese remarked that perhaps only a limited number of modules and certain types of molecular interactions could underlie the apparent complexity of signaling pathways and networks. This idea is both intuitively sound and intellectually attractive. It is supported by the fact that only a few intracellular modules, which mediate protein-protein interactions, namely the SH2, SH3, PH, PTB and WW domains, seem to be involved in an enormous multitude of membrane, cytoplasmic and nuclear processes in both unicellular and multicellular organisms. Moreover, a recent report from the Leder laboratory~ may indicate a new trend, namely that some of these modules could act in concert, adding another level of regulation and complexity to the existing pathways.

From SH3 to WW domain - a brief retrospect Based on their results with hostdependent mutants of the Rous sarcoma virus, in the late 1980s, Hirai and Varmus2 proposed that the amino-terminal regions of Src-family kinases form transient complexes with cellular proteins and that these complexes play an important role in the mechanism by which Src, Yes and other non-receptor-type kinases signal in normal and transformed cells. At that time, the SH2 domain residing at the amino-terminal half of the Src kinases had already been delineated, and was a primary candidate for a signaling module3. Shortly thereafter, the SH3 motif was defined and localized in Src just upstream of the SH2 sequence4. The Yes-associated protein (YAP) was isolated as one of the proteins that bind to the SH3 domain of Yes; its sequence contains an SH3-bin~lingmotif with the core consensus Px~P (where P = proline and x = any amino acid)s. It was through the detailed characterization of YAP that we obtained clues to a novel protein module6. This potential protein domain of 38 semi-conserved amino acids was identified by computeraided analysis of imperfectly repeated sequences in the mouse isoform of YAP, *Held at Mah~tea;.Ealy,between 7-12 May 1995. 9 1996,Elsevier Science Ltd

SH3 and WW bind similar ligands, but have different structures Using functional screens, two ligands that bind to the WW domain were identified 1~ Sequence comparison of the and also in a yeast factor Rsp5, a pro- ligands revealed a shared proline-rich tein with ubiquitin-ligase activity. As region that binds strongly and specifithree independent groups identified cally to the WW domain of human YAP. this motif, three different names were This region consists of the amino acid coined: the WW domain7, WWP motif8 sequence, PPPPY, which is perfectly and Rsp5-repeat 9. For the sake of sim- conserved between the two ligands. By plicity, we use here the term WW do- sequentially replacing each of these five main. The name refers to one of the positions with alanine for in vitro binddistinguishing features of the domain: ing assays, a preliminary consensus of the presence of two highly-conserved xPPxY was established for binding 1~ (L (a) Forrr

1

3P

Y A P Mutations

m YE~

BP

or

CRK

Polyproline

(d)

/

Dystroglycan u

ons

(e)

~ ~ 13APP

Dystrophi

FE65

N

Figure 1 Examples of the involvement of the WW domain-ligand link (the WW domain is in green, polyproline motifs are in blue and the SH3 domain is in magenta) in biological processes and genetic disorders, for example, in: (a) oncogenic transformation or viral budding, between the GAG-ONCS and Yes-associated proteins (YAPs); (b) Liddle's syndrome, between the 13-subunit of the epithelial sodium channel (i~ENaC) and WW; (c) limb and kidney development, between formin and formin-binding proteins (FBP); (d) muscular dystrophy, between dystrophin and dystroglycan; and (e) Alzheimer's disease, in the processing of the l~-amyloid precursor protein (13APP)and between the PTB domain and a motif (NPTY) that binds PTB of the FE65 factor. More detailed description of these molecular connections can be found in Ref. 12. Other abbreviations used: MA, modifying/processing activity directed to the 13-amyloid precursor proteins.

PII:S0968-0004(96)30018-2

161

FRONTLINES

.......

- -

TIBS 2 1 - MAY 1996

................

This consensus is distinct from the SH3binding motif PxxP. With these data, we implicated the WW domain in mediating protein-protein interactions, as a variant of the paradigm set by SH3 domains and their proline-rich ligands. Although the WW and SH3 domains are functionally similar, their structures are distinct. The preliminary nuclear magnetic resonance (NMR) structure of the WW domain of human YAP in complex with its cognate peptide suggests that the WW domain represents the most compact globular structure of 38 amino acids known to date (M. J. Macias et al., unpublished). It consists of a three-stranded antiparallel I~-sheet with a hydrophobic pocket composed of leucine, tyrosine and the second tryptophan. These amino acids are part of the ligand-binding interface, as deduced from structural and mutational studies (M. J. Macias et al., unpublished; M. Sudol and H. I. Chen, unpublished).

Emergingbiologyof the WW domain-ligand link To our surprise, in the last year the WW domain-ligand link has been

(a)

implicated, directly or indirectly, in several human genetic disorders 11 (Fig. 1). The best example comes from the study of Liddle's syndrome, a hereditary form of hypertension, which results from genetic lesions of the amiloride-sensitive epithelial sodium channel 12. The mutations that have been reported in patients with Liddle's syndrome all involve deletions that encompass a 12-residue sequence containing the PPPNY motif. Interestingly, two Liddle's syndrome patients were recently characterized with single amino acid substitutions: P616L (giving rise to PPLNY) and another with YB18H (giving rise to PPPNH) in the proline-rich motif 13,14. The 'alanine scan' of the proline-rich region of the sodium channel followed by a functional assay of the mutated channels in the Xenopus oocyte system definitively confirmed that the PPPNY motif behaves in the same way as the xPPxY consensus, which was established for a binder (the WBP-1 protein) to the WW domain of YAP (Refs 10, 15). As expected, one of the proteins containing the WW domain, namely Nedd4, was shown to bind specifically to the

(b)

PTYkinase j ]PTPPPLPP] I

l

I[

~

K'- 9 IPTPPPLPP] I

P"Z~I

e

!

II PTs PaseII

P~TYkinase" ~

PTSFFkinase

(c)

m §

PTYPase PTY kinase

Rgure 2 Prompted by the example of apparent degeneracy in signaling by WW and SH3 domains 1, we speculate here on the role of phosphorylation in disrupting or in generating new specificity in bindings between proline-rich ligands and various signaling modules. (a) Polyproline region of formins binds interchangeably to WW or SH3 domain of formin-binding proteins (FBPs). It is possible that phosphorylation of the threonine residue within the core sequence of the ligand (formins are indeed phosphoproteins) regulates this binding in a negative fashion. (b) The PTB domain was shown to interact with the phosphorylated or non-phosphorylated sequences with the NPxY core consensus. As NPxY is similar to PPxY (the core consensus for several binders to WW domains), we speculate that some WW domains may interact with PTB binders, perhaps at lower affinity. (c) The WW domain of Yes-associated proteins (YAP) binds a ligand with the core consensus xPPxY. It is tempting to speculate that phosphorytation of tyrosine residue within the core could represent a 'molecular switch' by which WW domain-ligand connection is replaced with SH2 domain-phosphorylated ligand link. Abbreviations used: PTY, protein tyrosine; PTS/T, protein serine/threonine; Pase, phosphatase; NPTY, a motif that in non-phosphorylated form binds to the PTB domain of FE65 factor. Phosphate is indicated by P in a green circle.

162

PPPNY-containing region of the wildtype sodium channel and that binding resulted in the suppression of the sodium channel activity ~4. Sequence comparison studies initially indicated the presence of the WW domain at the carboxy-terminai region of human dystrophin 7. More recently, using the yeast two-hybrid system and phage display libraries, we have identified potential ligands to the WW domain of dystrophin, among which is the 13-dystroglycan receptor, a protein known to be a part of the multi-component complex of dystrophin and that participates in communication between the extracellular matrix and the network of dystrophin-containing cytoskeleton (M. Sudol and C. Bougeret, unpublished). It is possible that mutations in the WW-binding domain of the [3-dystroglycan receptor might result in dystrophic phenotypes. The FE65 factor cloned and characterized by the Russo laboratory at the University of Naples contains one WW and two PTB domains 16. The PTB domains were shown to bind the 13-amyloid precursor protein (13APP)16. We have isolated a ligand to the WW domain of FE65, which may participate in the processing of the [3APP (M. Sudol, T. Russo and K. Ermekova, unpublished). Misprocessing of 13APP gives rise to the deposition of 13-amyloid in the brain, a process that correlates with the onset of Alzheimer's disease. The PPPPY motif was shown to be a functional 'domain' (also termed the 'L domain' for a Late function) present in retroviral GAG proteins and is identical to the core sequence of the binder to the WW domain of YAP]7. This precise five amino acid sequence is required for retroviral budding ]7. Retroviruses with the GAG protein in which the L domain is deleted do not bud at all. This observation raises the possibility that an interaction with a host protein that contains the WW domain is required for retroviral particles to pinch off from the cell surface. The L domain may also play a role in the neoplastic transformation by GAG-containing oncogenes (Fig. 1).

WW and SH3 act in concert? An intriguing report from the Leder laboratory proposed a functional interplay between the WW and SH3 domains 1. This hypothesis emerged from the study of formins, a set of protein isoforms encoded by the mouse limb deformity locus (ld) 18.As the first binders ever to the SH3 domain (of Abl) showed some similarity to the polyproline sequences in

FRONTLINES

TIBS 21 - MAY 1996

formins 19, it was suspected that formins may exert their function in limb and kidney development by interacting with SH3-containing proteins. An exhaustive screen of expression libraries from the mouse limb bud with the polyproline region of formin resulted in two sets of formin-binding proteins (FBPs); one set, as expected, has SH3 domains and the other has WW modules. Interestingly, the WW domains of FBPs were shown to compete with the SH3 domain of Abl in binding the polyproline peptide present in formin. The generalization from this example is that WW domains might regulate the function of SH3 domains by modulating their interaction with ligands through direct competition for the same proline-rich sequences on target proteins. This is fine proof of principle and an appealing scenario for a molecular mechanism regulating developmental events. If indeed these interactions are confirmed in vivo, they will provide one of the first examples of degeneracy in the 'protein-protein interaction code'.

WW is olderthan SH3 The WW domain-polyproline ligand link seems to be involved in many signaling pathways and may represent one of the basic molecular mechanisms used by adapter proteins to assemble signaling molecules. Unexpectedly, the WW module has been implicated in several human diseases - in sharp contrast, for example with the SH2 and SH3 domains, first described almost a decade ago. One possible explanation for this is that, as the WW domain seems to be evolutionarily older2~ - it is present in several proteins of yeast and also occurs in plants - Nature appears to have used this 'molecular adhesive' to solve basic protein-protein communication problems in 'primordial' pathways.

FromSH3 and WW to AA domains When the Yes proto-oncogene was isolated as the first Src-related gene, it was proposed that other Src-related genes exist. The emergence of the Src superfamily confirmed this proposal. By this imperfect analogy, we would like to suggest the existance of other protein modules composed of stacked aromatic (A) residues that have affinity for polyproline sequences. Perhaps, more competition is in store. References I Chan, 0. C., Bedford, M. T. and Leder, P. (1996) EMBO J. 15, 1045-1054 2 Hirai, H. and Varmus, H. (1990) Mol. Cell. Biol.

10, 1307-1318 3 Pawson, T. (1995) Nature 373, 573-580 4 Mayer, B. J., Hamaguchi, M. and Hanafusa, H. (1988) Nature 332, 272- 275 5 Sudol, M. (1994) Oncogene 9, 2145-2152 6 Sudol, M. et al. (1995) J. Biol. Chem. 270, 14733-14741 7 Bork, P. and Sudol, M. (1994) Trends Biochem. Sci. 19, 531-533 8 Andre, B. and Springael, J. Y. (1994) Biochem.

OKAy BO'r "f~ ~IcRoge A~:(I~LEk,A'Tofx I~ H~R~/

ACc~L~fo~?

Biophys. Res. Commun. 205,

1201-1205 9 Hofmann, K. and Bucher, P. (1995) FEBS Lett. 358, 153-157 10 Chen, H. I. and Sudol, M. (1995)

& ~ v ~ "~0 IJITM I~A~F

Proc. Natl Acad. Sci. USA 92, 7819-7823 11 Einbond, A. and Sudol, M. FEBS Lett.

(in press)

g~cT~IA I~T~ TflEI~I CoNSTI'FU.~NT ~ '

12 Rossier, B. C. (1996) Adv. Nephrol. 25,

275-286 13 Hansson, J. H. et al. (1995) Proc. Natl Acad. Sci. USA 92,

11495-11499 14 Staub, O. et aL EMBO J. (in press) 15 Snyder, P. M. et al. (1995) Cell 83,

969-978

P,,, NoT~t~k OTHr

16 Fiore, F. et al. (1995) J. Biol. Chem. 270,

30853-30856 17 Parent, L. J. et al. (1995) J. Virol. 69, 5455-5460 18 Woychik, R. P. et aL (1990) Nature 346, 850-853 19 Ren, R. et al. (1993) Science 259, 1157-1161 20 Sudol, M. et al. (1995) FEBS Lett. 369, 67-71

A~K US, A Irf,'~LJ'

J

~

]

,..

9tBrt-V Be 5 ~ m l / ~ 5

gy ~HAVE ~o F ~

- A(cor

"To MV S

MARIUS SUDOL Department of Biochemistry, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA. Email: M_Sudol@ smtplink.mssm.edu

~r:

T~ ~ t , g ~F I~I~- VI~L F-~E .,

~1"~1~' 163