The Structure of the talin head reveals a novel extended conformation of the FERM domain

FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains (F1, F2, F3) in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds β-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdIns(4,5)P2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state.     

The C terminus of initiation factor 4E-binding protein 1 contains multiple regulatory features that influence its function and phosphorylation

Eukaryotic initiation factor 4E (eIF4E) binds the mRNA cap structure and forms eIF4F complexes that recruit 40S subunits to the mRNA. Formation of eIF4F is blocked by eIF4E-binding proteins such as 4E-BP1, which interacts with eIF4E via a motif in the center of its 118-residue sequence. 4E-BP1 plays key roles in cell proliferation, growth, and survival. Binding of 4E-BP1 to eIF4E is regulated by hierarchical multisite phosphorylation. Here we demonstrate that three different features in the C terminus of 4E-BP1 play distinct roles in regulating its phosphorylation and function. Firstly, we identify a new phosphorylation site in its C terminus (S101). A serine or glutamate at this position is required for efficient phosphorylation at Ser65. A second C-terminal site, S112, directly affects binding of 4E-BP1 to eIF4E without influencing phosphorylation of other sites. Thirdly, a conserved C-terminal motif influences phosphorylation of multiple residues, including rapamycin-insensitive sites. These relatively long-range effects are surprising given the reportedly unstructured nature of 4E-BP1 and may imply that phosphorylation of 4E-BP1 and/or binding to eIF4E induces a more-ordered structure. 4E-BP2 and -3 lack phosphorylatable residues corresponding to both S101 and S112. However, in 4E-BP3, replacement of the alanine at the position corresponding to S112 by serine or glutamate did not confer the ability to be released from eIF4E in response to insulin.     

Expression and mutational analysis of Autographa californica nucleopolyhedrovirus HCF-1: functional requirements for cysteine residues

The host cell-specific factor 1 gene (hcf-1) of the baculovirus Autographa californica multiple nucleopolyhedrovirus is required for efficient virus growth in TN368 cells but is dispensable for virus replication in SF21 cells. However, the mechanism of action of hcf-1 is unknown. To begin to understand its function in virus replication we have investigated the expression and localization pattern of HCF-1 in infected cells. Analysis of virus-infected TN368 cells showed that hcf-1 is expressed at an early time in the virus life cycle, between 2 and 12 h postinfection, and localized the protein to punctate nuclear foci. Through coprecipitation experiments we have confirmed that HCF-1 self-associates into dimers or higher-order structures. We also found that overexpression of HCF-1 repressed expression from the hcf-1 promoter in transient reporter assays. Mutagenesis of cysteine residues within a putative RING finger domain in the amino acid sequence of HCF-1 abolished self-association activity and suggests that the RING domain may be involved in this protein-protein interaction. A different but overlapping set of cysteine residues were required for efficient gene repression activity. Functional analysis of HCF-1 mutants showed that the cysteine amino acids required for both self-association and gene repression activities of HCF-1 were also required for efficient late-gene expression and occlusion body formation in TN368 cells. Mutational analysis also identified essential charged and hydrophobic amino acids located between two of the essential cysteine residues. We propose that HCF-1 is a RING finger-containing protein whose activity requires HCF-1 self-association and gene repression activity.     

TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function

BACKGROUND: The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). We recently identified a TOR signaling (TOS) motif in the N terminus of S6K1 and the C terminus of 4E-BP1 and demonstrated that in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. RESULTS: Here, we show that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. CONCLUSIONS: Our data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth.