Laurence A. Lasky

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.

An endothelial ligand for L-Selectin is a novel mucin-like molecule

The adhesive interaction between circulating lymphocytes and the high endothelial venules (HEV) of lymph nodes (LN) is mediated by lymphocyte L-selectin, a member of the selectin family of cell adhesion proteins. Previous work has identified a sulfated 50 kd glycoprotein (Sgp50) as an HEV ligand for L-selectin. We now report the purification of this glycoprotein and the utilization of the derived N-terminal amino acid sequence to clone a cDNA. The predicted sequence reveals a novel, mucin-like molecule containing two serine/threonine-rich domains. The mRNA encoding this glycoprotein is preferentially expressed in LN. Antibodies against predicted peptides immunoprecipitate Sgp50 and stain the apical surface of LN HEV. These results thus define a tissue-specific mucin-like endothelial glycoprotein that appears to function as a scaffold that presents carbohydrates to the L-selectin lectin domain.

Cloning of a lymphocyte homing receptor reveals a lectin domain

Lymphocytes express cell surface molecules, termed homing receptors, that mediate their selective attachment to specialized high endothelial venules found within secondary lymphoid organs. Previous work has demonstrated that the adhesive interaction between lymphocytes and the endothelium of peripheral lymph nodes appears to involve a lectin-like activity. Moreover, MEL-14, a monoclonal antibody that blocks lymphocyte-peripheral lymph node binding and presumably recognizes the homing receptor mediating this adhesive interaction, appeared to detect the lectin-like receptor. In this paper we describe the cloning of a murine cDNA that encodes the antigen recognized by the MEL-14 antibody. Characterization of the cDNA encoding the putative mouse peripheral lymph node-specific homing receptor shows that it contains a lectin domain that appears to be involved in the binding of lymphocytes to peripheral lymph node endothelium, thus defining a new type of cellular adhesion molecule. This result supports a novel mechanism for the distribution of lymphocyte populations to various lymphoid organs.

AKT/PKB Phosphorylation of p21Cip/WAF1 Enhances Protein Stability of p21Cip/WAF1 and Promotes Cell Survival

p21Cip1/WAF1 (p21), a p53-inducible protein, is a critical regulator of cell cycle and cell survival. p21 binds to and inhibits both the DNA synthesis regulator proliferating cell nuclear antigen and cyclin A/E-CDK2 complexes. Recently, p21 has also been shown to be a positive regulator of cell cycle progression as p21 is necessary for the assembly and activation of cyclin D1-CDK4/6 complexes. Furthermore, elevated p21 protein levels have been observed in various aggressive tumors as well as linked to chemoresistance. Here we demonstrate that p21 is directly phosphorylated by AKT/PKB, a survival kinase that is hyperactivated in many late stage tumors. Two sites (Thr145 and Ser146) in the carboxyl terminus of p21 are phosphorylated by AKT/PKB in vitro and in vivo. Phosphorylation of Thr145 inhibits PCNA binding, whereas phosphorylation of Ser146 significantly increases p21 protein stability. Glioblastoma cell lines with activated AKT/PKB show enhanced p21 stability, and they are more resistant to taxol-mediated toxicity. Finally, AKT/PKB controls the assembly of cyclin D1-CDK4 complexes through modulation of p21 and cyclin D1 levels. These data imply that enhanced levels of p21 in tumors are due, in part, to phosphorylation by activated AKT/PKB. Furthermore, they suggest that one mechanism of AKT/PKB regulation of tumor cell survival and/or proliferation is to stabilize p21 protein.

A Specificity Map for the PDZ Domain Family

PDZ domains are protein–protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position −2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.

Selectins: a family of adhesion receptors.

Recent data have shown that a group of cell surface proteins, originally studied independently as lymphocyte homing receptors or as activation-induced surface proteins of platelets and/or endothelial cells (Stoolman, 1989) are structurally related. Each is an integral membrane protein with an N-terminal, C-type lectin domain followed by an EGF-like module, multiple copies of the consensus repeat units characteristic of complement-binding proteins, a transmembrane segment, and a short cytoplasmic domain. The three known proteins having this structure are encoded by closely linked genes on the long arm of human and mouse chromosome 1 (Watson et al., 1990). The gene structures are related, and the genes clearly arose by gene duplication. These proteins are all involved in cell-cell adhesion events and constitute a new family of cell adhesion receptors. A wide variety of names are used to designate these proteins, owing to their independent discovery by different laboratories working in several fields. This diversity of nomenclature interferes with the dissemination of information about these proteins. After consultation among the researchers working on these proteins and other scientists, we propose that this family of proteins be named selectins to reflect the involvement of carbohydrate recognition in their functions. Individual members of the family will be designated by a prefix capital letter, as is done for the cadherins (e. g., E-, N-, P-). Letters can be chosen based on the source of the original discovery but are not intended to imply cell type specificity. The three known selectins are: L-selectin

Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation.

The E6 proteins from the high-risk human papillomavirus (HPV) types have previously been shown to target a number of PDZ domain-containing proteins for proteasome-mediated degradation. These include the hDlg tumour suppressor and the MAGI-1 protein. In this study we show that high-risk HPV E6 proteins also target the related MAGI-2 and MAGI-3 proteins for degradation. Moreover, we show that the interaction is specific to one PDZ domain, and that co-expression of this domain can protect each of the full-length MAGI proteins from E6-mediated degradation. These data provide clear indicators for the potential design of compounds that could specifically inhibit the interaction of oncogenic HPV E6 proteins with an important class of target proteins.

Tyrosine phosphorylation regulates the SH3-mediated binding of the Wiskott-Aldrich syndrome protein to PSTPIP, a cytoskeletal-associated protein.

Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system. Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton. Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PSTPIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivoresults in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASPin vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton.

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain.

The LAP (leucine-rich repeatand PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW−4ETW−1V; IC50 = ∼0.15 μm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp−1 are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the β2-β3 loop provides a binding site for the Trp−4 side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.

Cytoskeletal Protein PSTPIP1 Directs the PEST-Type Protein Tyrosine Phosphatase to the c-Abl Kinase to Mediate Abl Dephosphorylation

A search for c-Abl interacting proteins resulted in the recovery of PSTPIP1, originally identified as a binding protein of the PEST-type protein tyrosine phosphatases (PTP). PSTPIP1 was phosphorylated by c-Abl, and growth factor-induced PSTPIP1 phosphorylation was diminished in Abl null fibroblasts. PSTPIP1 was able to bridge c-Abl to the PEST-type PTPs. Several experiments suggest that the PEST-type PTPs negatively regulate c-Abl activity: c-Abl was hyperphosphorylated in PTP-PEST-deficient cells; disruption of the c-Abl-PSTPIP1-PEST-type PTP ternary complex by overexpression of PSTPIP1 mutants increased c-Abl phosphotyrosine content; and PDGF-induced c-Abl kinase activation was prolonged in PTP-PEST-deficient cells. Dephosphorylation of c-Abl by PEST-type PTP represents a novel mechanism by which c-Abl activity is regulated.