Brian K. Kay

The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

A common focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two‐hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to ones favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3‐dimensional analyses of antigen‐antibody interactions have revealed that epitopes are typically 4–7 residues long (1). Other examples include protein‐interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc‐large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14–3–3 proteins that typically recognize linear regions of 3–9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2–7). Among the primary structures of many ligands for protein–protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein‐interaction domains prefer ligand sequences that are proline‐rich. In addition, even though ligands for EH domains and 14–3–3 domains are not proline‐rich, they do include a single proline residue. This review highlights the analysis of those protein‐protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.—Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline‐rich motifs in signaling proteins with their cognate domains. FASEB J. 14, 231–241 (2000)

Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

Intersectin, a Novel Adaptor Protein with Two Eps15 Homology and Five Src Homology 3 Domains

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX 1–2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.

A novel, multifunctional c-Cbl binding protein in insulin receptor signaling in 3T3-L1 adipocytes

ABSTRACT The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.

Signaling on the Endocytic Pathway

Ligand binding to receptor tyrosine kinases and G‐protein‐coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin‐coated pits and vesicles. While receptor‐mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand‐activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal‐regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin‐dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.

An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets

We have examined the potential of isolating novel ligands from a library of M13 pIII-fusion phage displaying peptides composed of 38 random amino acids (aa). The library was panned with streptavidin (SA) and a polyclonal goat antimouse IgG Fc antibody (Ab) preparation coupled to paramagnetic beads. SA selected two classes of phage from the library. One class exhibited the aa motif, HP(Q/M) theta (where theta signifies a non-polar aa), similar to the motif identified by Devlin et al. [Science 249 (1990) 404-406] using a 15-aa random peptide library displayed on phage. The other class of phage had no discernible motif. In binding experiments, the non-HP(Q/M) theta phage had a slightly higher affinity for SA than did the motif phage. Both classes of SA-binding phage failed to bind native and non-glycosylated forms of avidin, even though SA and avidin are structurally similar and both proteins possess extraordinary affinities for biotin. The polyclonal goat anti-mouse IgG Fc Ab preparation selected phage displaying sequences similar to a region of the mouse IgG Fc. Thus, a single immunodominant epitope on the mouse IgG Fc was identified. Furthermore, a second phage displaying peptides with no discernible sequence similarities to mouse IgG Fc was isolated. Thus, an M13 library displaying 38-aa peptides can yield phage with affinity for various targets. Finally, we have observed a biological bias against odd numbers of Cys residues in the displayed peptides.

Splice Variants of Intersectin Are Components of the Endocytic Machinery in Neurons and Nonneuronal Cells

We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

Characterization of phage that bind plastic from phage-displayed random peptide libraries

During routine screenings of random peptide libraries displayed at the N terminus of the pIII coat protein of M13 bacteriophage, clones were isolated that bound directly to the polystyrene (PS) surface used to immobilize the target protein. The plastic-binding phage (P-b phi) bind to both unblocked plastic (PS and polyvinyl chloride, PVC) and plastic blocked with bovine serum albumin (BSA) but require non-ionic detergent to bind to plastic blocked with milk. Comparison of the P-b phi to antibody-binding phage (Ab-b phi) indicates that similar numbers of phage particles are bound, but fewer P-b phi the recovered by acid elution. Sequence determination of the displayed peptides reveals they lack amino-acid sequence similarity yet are highly enriched for the Tyr and Trp residues. However, because not all phage that display peptides rich in Tyr and Trp residues bind to plastic, and other methods of screening random peptide libraries have identified different classes of plastic-binding peptides, the relative abundance of Tyr and Trp residues should not be considered diagnostic of plastic-binding. In summary, these results help characterize one of the most common methods used to screen random peptide libraries and suggest strategies to avoid isolating P-b phi. Furthermore, while it is generally believed that proteins bind to plastic by non-specific interactions, these results show that a bias in aa composition can exist.

Isolation of a NCK-associated kinase, PRK2, an SH3-binding protein and potential effector of Rho protein signaling

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cγ. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling.