Large metal ions (>0.9 A ionic radius) have previously been found to bind only weakly to human serum transferrin (hTF, 80 kDa), presumably because the interdomain cleft cannot close around the metal and synergistic anion. Surprisingly, therefore, we report that Bi3+ (ionic radius 1.03 A), a metal ion widely used in anti-ulcer drugs, binds strongly to both the N- and C-lobes with log K1* = 19.42 and log K2* = 18.58 (10 mM Hepes, 5 mM bicarbonate, 310 K). The uptake of Bi3+ by apo-hTF from bismuth citrate complexes is very slow (hours), whereas that from bismuth nitrilotriacetate is rapid (minutes). Evidence from absorption and NMR spectroscopy is presented to show that Bi3+ binds to the specific Fe3+ binding sites along with carbonate as the synergistic anion. Under the conditions used, preferential binding of Bi3+ to the C-lobe of hTF is observed. Linear free energy relationships show that there is a strong correlation between the strength of binding of Bi3+ and Fe3+ to a wide variety of ligands which include transferrin. Therefore we conclude that the strength of metal ion binding to transferrin is determined more by the ligand donor set than by the size of the ion.

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Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These (14) used a synthetic peptide library to identify Src and P13K SH3 ligands with the consensus sequence RXLPPZP (Z = L for Src and R for P13K) and demonstrated that each SH3 domain bound its respective ligand with higher affinity than did the other.In an effort to develop a more detailed understanding of the specificity of SH3-ligand interactions, we have constructed a phage-displayed library (termed the PXXP library) encoding peptides of the form X6PXXPX6, where X represents any of the 20 naturally occurring amino acids and P represents invariant proline residues. Using this library, we have identified peptide ligands for SH3 domains of Src, Yes, Abl, Cortactin, p53bp2, PLCy, Crk, and Grb2. Each SH3 domain selects a set of peptide ligands sharing a distinct consensus motif; these motifs reflect the unique ligand preferences of each SH3 domain. MATERIALS AND METHODSPreparation of Glutathione S-Transferase (GST)-SH3 Fusion Proteins. Constructs encoding GST fusions to the Grb2 N-terminal (Grb2 N, aa 1-58), Grb2 C-terminal (Grb2 C, aa 154-217), Nck N-terminal (Nck N, aa 1-68), Nck middle (Nck M, aa 101-166), Nck C-terminal (Nck C, aa 191-257), p53bp2 (aa 454-530), or Src (aa 87-143) SH3 domains were generated by PCR cloning of the appropriate cDNAs into pGEX-2T (Pharmacia). The integrity of the constructs was confirmed by DNA sequencing. pGEX-derived constructs expressing GST fusions to the SH3 domains of Yes, Cortactin, Crk, Abl, and PLC,y were kindly provided by M. Sudol (Rockefeller University), J. T. Parsons (University of Virginia), M. Matsuda

Recently the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product has been identified as a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, we examined whether a Ras/Rap-related GTPase might be involved in this process. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promoted the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation was also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug.The Ras subfamily of small GTPases regulates a vast array of biological events that include cell growth, differentiation, and transformation (1). The prototypic Ras proteins, Ha-, K-, and N-Ras, are known to transduce mitogenic and differentiation signals from cell surface receptors to the nucleus (1). However, despite their conservation throughout eukaryotic evolution, the function of Ras-related GTPases such as Rap1, R-Ras, Ral, Rheb, and Rit remains, at best, poorly understood. Ras activity is regulated by a GDP/GTP cycle whereby guanine nucleotide exchange factors promote the release of GDP from inactive Ras, facilitating its loading with the more abundant GTP (2). Binding to GTP induces a conformational change enabling interaction with and activation of downstream effector proteins (1). The termination of this signal is regulated by GTPase-activating proteins (GAPs), 1 which greatly accelerate the intrinsic GTPase activity of Ras, returning it to its inactive state (2). Loss of Ras GAP, as occurs in the genetic disorder neurofibromatosis type 1, results in increased Ras GTP levels, which contribute to tumor development (3, 4).Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are additional diseases that could be linked to the loss of GTPase regulation. TSC is a genetic disorder that results in the formation of benign tumors known as hamartomas, most typically found in kidney, brain, heart, and lung (3,5). LAM is a devastating lung disease that affects mainly women and is characterized by proliferation of atypical smooth muscle cells within the lung parenchyma (6). The inactivation of two tumor suppressor genes has been associated with TSC and LAM: TSC1 encodes the protein hamartin (TSC1), and TSC2 that encodes tuberin (TSC2) (7-9). Although TSC1 contains coiled-coil domains that are important for the formation of a functional complex with TSC2, TSC2 shares sequence homology with a family of GAPs that regulate the Ras-related GTPase, Rap1 (5). Accordingly, TSC2 has been reported to increase the intrinsic GTPase activity of Rap1 and also Rab5 in vitro (10, 11). However, it is not known whether this activity occurs in vivo or if it contributes to the physiological function of the TSC1-TSC2 complex.Several recent studies...

The p21-activated kinases (PAKs) link G protein-coupled receptors and growth factor receptors (S. Dharmawardhane, R. H. Daniels, and G. M. Bokoch, submitted for publication) to activation of MAP kinase cascades and to cytoskeletal reorganization (M. A. Sells, U. G. Knaus, D. Ambrose, S. Bagrodia, G. M. Bokoch, and J. Chernoff, submitted for publication). The proteins that interact with PAK to mediate its cellular effects and to couple it to upstream receptors are unknown. We describe here a specific interaction of the Nck adapter molecule with PAK1 both in vitro and in vivo. PAK1 and Nck associate in COS-7 and Swiss 3T3 cells constitutively, but this interaction is strengthened upon platelet-derived growth factor receptor stimulation. We show that Nck binds to PAK1 through its second Src homology 3 (SH3) domain, while PAK1 interacts with Nck via the first proline-rich SH3 binding motif at its amino terminus. The interaction of active PAK1 with Nck leads to the phosphorylation of Nck at multiple sites. Association of Nck with PAK1 may serve to link this important regulatory kinase to cell activation by growth factor receptors.

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Dynamic remodeling of spiny synapses is crucial for cortical circuit development, refinement, and plasticity, while abnormal morphogenesis is associated with neuropsychiatric disorders. Here we show in cultured rat cortical neurons that activation of Epac2, a PKA-independent cAMP target and Rap guanine-nucleotide exchange factor (GEF), induces spine shrinkage, increases spine motility, removes synaptic GluR2/3-containing AMPA receptors, and depresses excitatory transmission, while its inhibition promotes spine enlargement and stabilization. Epac2 is required for dopamine D1-like receptor-dependent spine shrinkage and GluR2 removal from spines. Epac2 interaction with neuroligin promotes its membrane recruitment and enhances its GEF activity. Rare missense mutations in the EPAC2 gene, previously found in individuals with autism, affect basal and neuroligin-stimulated GEF activity, dendritic Rap signaling, synaptic protein distribution, and spine morphology. Thus, we identify a novel mechanism that promotes dynamic remodeling and depression of spiny synapses, whose mutations may contribute to some aspects of disease.

The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.