Probing WW Domains to Uncover and Refine Determinants of Specificity in Ligand Recognition.

Understanding the specificity of protein-protein interaction mediated by domains and their ligands will have strong impact on basic and applied research. Visual inspection of WW domain sequences prompted a general classification of the domains into two large subfamilies. One subfamily contains two consecutive aromatic residues in the beta 2 strand of the domain whereas the other contains three or four consecutive aromatic residues in the same position. In the recent past, we proposed a rule of 'two vs. three aromatics' in the beta 2 strand of WW domains as a molecular discriminator between Class I and Class II WW domains, which recognize PPxY or PPLP motifs, respectively. Using phage display libraries expressing WW domains with random sequences replacing a part of the beta 2 strand, we provided additional evidence supporting our rule. We conclude that three consecutive aromatic amino acids within the beta 2 strand of WW domain are required but not always sufficient for the WW domain to belong to Class II.

Pulling strings below the surface: hormone receptor signaling through inhibition of protein tyrosine phosphatases.

Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptor's tyrosine residues. At any given moment, the receptor's phosphorylation state depends on the balance of kinase and phosphatase activities. Recent findings point to the exciting possibility that receptor signaling can be regulated by inhibition of protein tyrosine phosphatases (PTPs) that specifically hydrolyze receptor tyrosine-phosphates, or their immediate downstream effectors. This strategy has now been firmly validated for the insulin receptor and PTP1B; inhibiting PTP1B activity results in stimulation of the insulin receptor and signaling, even in the absence of insulin. This and similar findings suggest that PTP inhibitors have potential as hormone mimetics. In the present review, we outline this new paradigm for therapeutic regulation of the insulin receptor and discuss evidence that hints at other specific receptor-PTP pairs.

Yes-associated protein and p53-binding protein-2 interact through their WW and SH3 domains.

To understand the role of the Yes-associated protein (YAP), binding partners of its WW1 domain were isolated by a yeast two-hybrid screen. One of the interacting proteins was identified as p53-binding protein-2 (p53BP-2). YAP and p53BP-2 interacted in vitro and in vivo using their WW1 and SH3 domains, respectively. The YAP WW1 domain bound to the YPPPPY motif of p53BP-2, whereas the p53BP-2 SH3 domain interacted with the VPMRLR sequence of YAP, which is different from other known SH3 domain-binding motifs. By mutagenesis, we showed that this unusual SH3 domain interaction was due to the presence of three consecutive tryptophans located within the betaC strand of the SH3 domain. A point mutation within this triplet, W976R, restored the binding selectivity to the general consensus sequence for SH3 domains, the PXXP motif. A constitutively active form of c-Yes was observed to decrease the binding affinity between YAP and p53BP-2 using chloramphenicol acetyltransferase/enzyme-linked immunosorbent assay, whereas the overexpression of c-Yes did not modify this interaction. Since overexpression of an activated form of c-Yes resulted in tyrosine phosphorylation of p53BP-2, we propose that the p53BP-2 phosphorylation, possibly in the WW1 domain-binding motif, might negatively regulate the YAP.p53BP-2 complex.

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II.

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.

The WW domain of dystrophin requires EF-hands region to interact with beta-dystroglycan.

Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein beta-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with beta-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of beta-dystroglycan. This region of dystrophin implicated in binding beta-dystroglycan contains four modular protein domains: a WW domain, two putative Ca2+-binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38-40 amino acids with two highly conserved tryptophan residues spaced 20-22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding beta-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of beta-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxy-terminus of beta-dystroglycan occurs via a beta-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/beta-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.

A single point mutation in a group I WW domain shifts its specificity to that of group II WW domains.

WW domains can be divided into three groups based on their binding specificity. By random mutagenesis, we switched the specificity of the Yes-associated protein (YAP) WW1 domain, a Group I WW domain, to that of the FE65 WW domain, which belongs to Group II. We showed that a single mutation, leucine 190 (betaB5) to tryptophan, is required to switch from Group I to Group II. Although this single substitution in YAP WW1 domain is sufficient to precipitate the two protein isoforms of Mena, an in vivo ligand of FE65, we showed that an additional substitution, histidine 192 (betaB7) to glycine, significantly increased the ability of YAP to mimic FE65. This double mutant (L190W/H192G) precipitates eight of the nine protein bands that FE65 pulls down from rat brain protein lysates. Based on both our data and a sequence comparison between Group I and Group II WW domains, we propose that a block of three consecutive aromatic amino acids within the second beta-sheet of the domain is required, but not always sufficient, for a WW domain to belong to Group II. These data deepen our understanding of WW domain binding specificity and provide a basis for the rational design of modified WW domains with potential therapeutic applications.

Regulation of cdc2 gene expression by the upstream stimulatory factors (USFs).

cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been reported to play a central role, they cannot account for all aspects of the fine modulation of cdc2 gene expression during cell cycle and embryonic development. To characterize the transcription factors that control cdc2 gene expression during nerve cell differentiation in avians, we have previously cloned the quail cdc2 gene promoter region. We had identified an octamer (CAGGTGGC) containing an E-box, which has important activity in regulating cdc2 transcription. Using in vivo genomic footprinting experiments, we show here that this motif, currently named IG, is the target of binding proteins at different stages of neuroretina development, confirming its importance as a regulatory response element for cdc2 gene expression. A subset of Helix-Loop-Helix family of transcription factors, known as Upstream Stimulatory Factors (USFs) specifically bind to this sequence as dimers. Moreover, our results indicate that USFs transactivate the promoter of cdc2 via the IG motif. These data may help to better understand the mechanisms that control cell division in differentiating nerve cells.

Transient accumulation of retinoblastoma/E2F-1 protein complexes correlates with the onset of neuronal differentiation in the developing quail neural retina.

Retinoblastoma (Rb) protein has been implicated in the control of cell proliferation and malignant transformation in different cell types. To analyze its role as a promoter of cell growth arrest during development, we have studied the temporal pattern of Rb expression and its association with E2F-1 during embryogenesis of the quail neuroretina. During development of this neural organ, most cells stop dividing and begin to differentiate at embryonic days E6 and E7, as indicated by the decline of cyclin-dependent kinase cdk2 and by an increased level of cdk5. At this stage, we observed a shift of hyperphosphorylated Rb protein to its hypophosphorylated form as well as a decrease of the total level of Rb. The Rb-related protein, p107, is also progressively down-regulated from the E7 stage onwards. P130 levels, on the other hand, actually increase. Moreover, cell cycle exit at E6-E7 is characterized by a sudden and transient rise of the E2F-1/RB complex followed by the appearance of the E2F-4/p130 complex starting at E8. Conversely, expression of adenovirus E1A protein in E6 neuroretina cells leads to a dissociation of E2F-1/Rb complex and suppression of cell growth arrest and differentiation. This suggests that cell cycle exit and re-entry may depend on Rb/E2F-1 interaction. Although the rate of Rb synthesis declines in postmitotic cells, as suggested by in vivo metabolic labeling of the Rb protein, the level of the Rb transcript remains

Regulation of E2F-1 gene expression in avian cells.

E2F-1 is the prototype of a family of transcription factors playing a central role in the control of cell proliferation and apoptosis. E2F DNA binding activity is down-regulated during cellular differentiation, which is correlated with cell division arrest. We report here that the expression of E2F-1 itself is down-regulated in the developing quail neural retina between embryonic days E8-E10. This event occurs just after the massive arrest of the quail neuroretina cell division (E7-E8). To gain further insight into the regulatory mechanisms monitoring E2F-1 expression in differentiating neurons, we have cloned the quail E2F-1 promoter. In vivo DNA footprintings of this promoter have shown that a number of potential SP-1 and C/EBP response elements are constitutively occupied in the entire quail neuroretina of E5 and E14, whereas the two consensus palindromic E2F binding sites are only protected at E5. This suggests that these E2F elements participate in down-regulation of E2F-1 gene expression during avian neuroretina development. CAT reporter assays have shown that E2F-1 in association with its partner DP-1 transactivates its own promoter, whereas p105Rb inhibits the E2F-1 promoter. Both E2F-1/DP-1 and p105Rh require the presence of the E2F binding sites to mediate their effects. However, experiments performed with deletion mutants of the promoter strongly suggest that other regions located upstream of the E2F binding sites also mediate part of the E2F-1 transactivating effect on its own promoter. Altogether, these results suggest that the down-regulation of E2F-1 gene expression in differentiating neurons could be due, in part, to the E2F/Rb complexes binding to the E2F-1 promoter.

p34(cdc2) and mitotic cyclin expression in the developing quail neuroretina.

After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukaryotes remain postmitotic during their entire lifespan. This requires that a very stringent control be exerted on the cell division apparatus, whose molecular mechanisms remain quite elusive. Here we have used quail neuroretina as a model to study the control of cell division in the developing CNS. In vertebrates, embryonic neuroretinal cells (NR cells) stop their proliferation at different times depending on the cell type. Most NR cells in the quail embryo become postmitotic between E7 and E8. To acquire a better understanding of the molecular events leading to quiescence in NR cells, we have analyzed the expression of cdc2 and of two activators of p34(cdc2): cyclin A and cyclin B2 in the developing neuroretina. We report that these three proteins are downregulated between E7 and E9, suggesting that a common mechanism could block their transcription in differentiating neurons. We also report, using an immunohistochemical approach, that p34(cdc2) downregulation is correlated with the appearance of the microtubule-associated protein tau. These results strongly suggest that inhibition of cdc2 gene expression is closely linked to the achievement of terminal differentiation in neurons. However, we also show that postmitotic ganglion cells precursors begin to synthesize the early neuronal differentiation marker beta3-tubulin while p34(cdc2) is still detectable in these cells, suggesting that p34(cdc2) or a closely related kinase could play a role in some "young" postmitotic neurons.

Two distinct regulatory elements control quail cdc2 transcription: possible involvement in the control of retinoblast differentiation.

It is a characteristic of the central nervous system of higher eukaryotes that neurons, after an initial proliferation phase, remain postmitotic for their whole life span. In the developing quail neuroretina, most retinoblasts become postmitotic after 7-8 days of incubation. They also cease to express cdc2, which is presumably necessary to allow retinoblasts to definitively leave the cell cycle. The molecular mechanisms monitoring cdc2 expression during differentiation remain partly understood. To further study the control of cdc2 transcription in avian cells, we have cloned the quail cdc2 promoter. Two functional regulatory elements have been characterized. One of them contains an E2F-binding site. Human E2F-1 was found to transactivate the quail cdc2 promoter very efficiently in avian and human cells. Gel retardation experiments are presented, suggesting that E2F, in association with different partners, is a major regulatory of cdc2 transcription during the development of the neuroretina. Our data also indicate that another transcription factor binds to the octamer CAGGTGGC located 115 nucleotides above the main transcription start site. This motif is thus another important regulatory element participating in the control of cdc2 expression.