We have identified, characterized and cloned human, mouse and chicken cDNA of a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. We subsequently named it YAP for Yes-associated protein. Analysis of the YAP sequence revealed a protein module that was found in various structural, regulatory and signaling molecules. Because one of the prominent features of this sequence motif is the presence of two conserved tryptophans (W), we named it the WW domain. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous proline-rich region. Binding assays and site-specific mutagenesis have shown that the proline-rich motif binds with relatively high affinity and specificity to the WW domain of YAP, with a preliminary consensus that is different from the SH3-binding PXXP motif. This suggests that the WW domain has a role in mediating protein-protein interactions via proline-rich regions, similar but distinct from Src homology 3 (SH3) domains. Based on this finding, we hypothesize that additional protein modules exist and that they could be isolated using proline-rich peptides as functional probes.Key words: Protein protein interaction; Protein module; Polyproline Background and rationale -Retrospective lookWhat started as a pilot project ended up being the main focus of an entire lab. Our general aim has been to decipher molecular steps of signaling by the Yes proto-oncogene product which represents a non-receptor type protein-tyrosine kinase of the Src family [1]. The specific goal was to isolate substrates and regulators of the Yes kinase in order to understand at least some aspects of the molecular role it plays in normal and the viral-Yes oncogene transformed cells [2]. Initially, our experimental approaches were descriptive of nature [3,4]. We argued that by characterizing the pattern of Yes expression in various tissues and cells, we would have been able to find a common denominator that would provide a clue regarding the physiological function of the Yes protein [5]. Localization of the Yes proto-oncogene transcript and protein in cerebellar Purkinje cells was an exciting finding which gave us hope for functional clues [6]. Unfortunately, that was immediately followed by the frustration of trying to study the Yes kinase in difficult experimental systems of isolated Purkinje neurons or cerebellar slices. The direction of our research activities shifted swiftly when Hirai and Varmus proposed that the amino termini of Src family kinases form complexes with cellular proteins and that these apparently transient and dynamic complexes constitute a part of the mechanism by which Src, Yes and other kinases signal [7]. The SH2 domain, residing at the amino terminal half of the Src kinases had already been delineated at that time and was a primary candidate for a signaling domain [8]. The proposal of Hirai...

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6–Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.

Various cellular functions such as cell motility, cell survival, cytokinesis, and neurite outgrowth are dependent on temporal and spatial reorganization of the actin cytoskeleton. Rearrangement of the actin cytoskeleton results from signals activated by soluble factors (inside-out signals) and cell-substratum and cell-cell adhesion molecules (outside-in signals). In cultured cells, integration of these signals takes place in the focal adhesions (58) which are associated with well-defined actin stress fibers and provide tight binding to the underlying extracellular matrix. These contractile stress fibers are postulated to exert tension on the substratum and to play a role in morphogenesis and regulate cell motility.Actin stress fibers and focal adhesions form in quiescent fibroblasts in response to microinjection of constitutively active Rho GTPase or by extracellular signals such as lysophosphatidic acid (LPA) and bombesin (62) which lead to the activation of Rho. ADP-ribosylation and inhibition of Rho by Clostridium botulinum C3 toxin prevent this process. During focal adhesion assembly, several adhesion-associated proteins, including focal adhesion kinase (FAK), paxillin, and p130 cas , become tyrosine phosphorylated, suggesting the involvement of a tyrosine phosphorylation cascade in this event (9,65,77). LPA-induced activation of Rho can be blocked by an inhibitor of tyrosine kinase signaling, tryphostin, suggesting that tyrosine kinases act upstream of Rho activation (57). However, it has also been demonstrated that introduction of activated Rho into cells induces tyrosine phosphorylation of FAK, paxillin, and p130cas , placing tyrosine kinases downstream of Rho (18). To further support this concept, another tyrosine kinase inhibitor, genistein, prevents the formation of stress fibers after microinjection of constitutively active Rho (61). Besides serum-derived factors, binding of integrins to extracellular matrix proteins also activates Rho and induces stress fiber formation in the absence of serum (4). It is currently not well understood how integrin engagement, tyrosine kinase signaling, Rho activation, and formation of focal adhesions are integrated and coordinated at the cell membrane.According to a recent model, intracellular components of the focal adhesion complex and actin filaments associate with integrins upon integrin engagement with the extracellular matrix. In the presence of active Rho, these complexes and actin cluster by acto-myosin contraction, which leads to focal adhe-* Corresponding author. Mailing address: The Rockefeller University,