To control agonist-induced nuclear translocation of transcription factor kappa B (NF-kappa B) in intact cells, cell-permeable synthetic peptides were devised. Their import into intact cells was dependent on a hydrophobic region selected from the signal peptide sequences and was verified by their inaccessibility to extracellular proteases and by confocal laser scanning microscopy. When a cell-permeable peptide carried a functional cargo representing the nuclear localization sequence of NF-kappa B p50, it inhibited in a concentration-dependent manner nuclear translocation of NF-kappa B in cultured endothelial and monocytic cells stimulated with lipopolysaccharide or tumor necrosis factor-alpha. Synthetic peptide analogues with deleted hydrophobic cell membrane-permeable motif or with a mutated nuclear localization sequence were inactive. Cell membrane-permeable peptides were not cytotoxic within the concentration range used in these experiments. These results suggest that cell-permeable synthetic peptides carrying a functional cargo can be applied to control signal transduction-dependent subcellular traffic of transcription factors mediating the cellular responses to different agonists. Moreover, this approach can be used to study other intracellular processes involving proteins with functionally distinct domains.

Epidermal growth factor (EGF)-stimulated Ras activation involves specific interactions between the EGF receptor (EGFR), the adaptor proteins Grb2 and Shc, and the nucleotide exchange factor Sos-1. Study and control of these protein-protein interactions in vivo can be greatly promoted by introducing intracellular reagents that mimic EGFR functions. Here, we showed that a synthetic phosphopeptide encompassing the autophosphorylation site 1068 of EGFR formed a complex with endogenous Grb2 after this peptide was delivered into intact cells by a cell-permeable peptide import technique. Consequently, this intracellular peptide inhibited EGF-induced EGFR/Grb2 associations but not EGFR/Shc or Shc/Grb2 associations. Peptide-mediated disruption of the EGF/Grb2/Sos-1 cascade led to reduced Ras activation and mitogen-activated protein kinase activation. These results indicate that the binding of Grb2 to the phosphorylated Tyr-1068 of EGFR is crucial to the EGF-induced Ras/mitogen-activated protein kinase signaling pathway. The application of cell-permeable peptides to this study demonstrates a useful biochemical tool to probe and control various intracellular processes involved in signal transduction and gene transcription.Mitogenic signaling stimulated by epidermal growth factor (EGF) 1 requires the intrinsic tyrosine kinase activity of its transmembrane receptor (for review see Refs. 1 and 2). A number of intracellular proteins including the EGF receptor itself are phosphorylated on tyrosine residues in EGF-stimulated cells. Several autophosphorylation sites have been identified in the carboxyl-terminal region of the EGF receptor. These sites and their flanking regions constitute specific motifs that can be recognized by the Src homology 2 (SH2) domains or the phosphotyrosine binding/phosphotyrosine interaction domains of many intracellular signaling proteins (for review see Refs. 3 and 4).Ras activation originating from the EGF receptor comprises a cascade of protein-protein interactions involving a Grb2/Sos-1 signaling protein complex (5-12). The Grb2 protein, which functions as an adaptor, binds to tyrosine-phosphorylated EGF receptor either directly by its SH2 domain or indirectly via the Shc protein (13). Recent studies using in vitro peptide competition assays and EGFR mutants have suggested that the phosphotyrosine (Tyr(P)) 1068 of activated EGFR is a direct binding site for the Grb2 SH2 domain (11, 14 -17). To determine the functional consequence of disrupting the EGFR/Grb2 proteinprotein association in vivo, we delivered a peptide mimicking this EGFR autophosphorylation site into intact cells by using a nondestructive cell-permeable peptide import method (see Ref. 18). We showed in this report that this outside-in peptide formed an intracellular peptide-protein complex with endogenous Grb2, resulting in a substantial inhibition of EGF-stimulated EGFR/Grb2 association and Ras/MAP kinase activation. Our results demonstrate that intracellular tyrosine kinase signaling pathways can be studied and regulated in living...

The discovery of methods for generating proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. We report a method to engineer proteins with cell-membrane permeability. After a 12-amino acid residue membrane-translocating sequence (MTS) was fused to the C-terminus of glutathione S-transferase (GST), the resultant GST-MTS fusion proteins were efficiently imported into NIH 3T3 fibroblasts and other cells. To explore the applicability of this nondestructive import method to the study of intracellular processes, a 41-kDa GST-Grb2SH2-MTS fusion protein containing the Grb2 SH2 domain was tested for its effect on the epidermal growth factor (EGF)-stimulated signaling pathway. This fusion protein entered cells, formed a complex with phosphorylated EGF receptor (EGFR), and inhibited EGF-induced EGFR-Grb2 association and mitogen-activated protein kinase activation.

Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin j33 (glycoprotein IIIa) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin j33 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin (83 heterodimers, "ubP3, and "I33, respectively. Inhibition of adhesion was integrin-specific because the cellpermeable f33 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin j81 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin 61 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL (8, 9). A structure-function analysis of the cytoplasmic segment of integrin P3 is needed to pinpoint its regulatory sites.We undertook analysis of the 41-residue cytoplasmic tail of integrin 3 by applying our recently developed cell-permeable peptide import technique (10) to probe integrin 3 cytoplasmic protein-protein interactions. As a functional endpoint, we used adhesion of human erythroleukemia (HEL) cells to immobilized fibrinogen in response to stimulation with 4(3-phorbol 12-myristate 13-acetate (PMA). HEL cells express endogenous integrin alIIb3 and serve as a useful model for structure-function studies of platelet constituents (11,12). The integrin 3 is also expressed as a heterodimer with integrin av in human platelets and endothelial cells (13). Therefore, we studied adhesion of the ECV 304 cell line derived from human umbilical vein endothelial cells that express aCvP3 integrin (vitronectin receptor) (14). Using cell-permeable peptides representing wild-type and mutated sequences, we have identified the major cell adhesion regulatory domain (CARD) of integrin 3. It encompasses a 16-amino acid sequence of its cytoplasmic tail. A synthetic peptide mimetic representing CARD imported by HEL and ECV 304 cells inhibits "from within" their adhesion to immobilized fibrinogen by competing with intracellular protein-protein interactions involving the integrin (3 cytoplasmic tail. MATERIALS AND METHODSSynthetic Peptides, Antibodies, and Cell Lines. Peptides were synthesized by a step-wise solid-phase peptide synthesis method and purified by C18 reverse-phase high performance liquid chromatography (HPLC) (10). As depicted in Fig. 1, overlapping peptides encompassing the entire integrin (3 cytoplasmic sequence (6, 7) represent residues 722-737 (peptide (33-3), 735-750 (peptide (33-2), 747-762 (peptide (33-1), and 742-755 (peptide (33-4). The cell-permeable ...

Fibroblast growth factor-1 (FGF-1) is a potent mitogen for mesoderm-and neuroectoderm-derived cell types in vitro. However, a mutant FGF-1 with deletion in its nuclear localization sequence (NLS, residues 21-27) is not mitogenic in vitro. We demonstrated that synthetic peptides containing this NLS were able to stimulate DNA synthesis in a FGF receptor-independent manner after they were delivered into living NIH 3T3 cells by a cellpermeable peptide import technique. The stimulation of maximal DNA synthesis by these peptides required the presence of peptides during the entire G 1 phase of the cell cycle. The mitogenic effect was specific for the NLS of FGF-1 because a peptide with double point mutations at lysine residues was inactive in stimulating DNA synthesis. Our results suggest that the NLS plays an important role in the mitogenic pathway initiated by exogenous FGF-1 by its direct involvement in the nuclear transport and signaling of internalized FGF-1.Mitogenic signaling for many growth factors is triggered by their binding to the transmembrane receptor tyrosine kinases, for example, those for epidermal growth factor and plateletderived growth factor. Upon ligand-receptor binding, these receptors are dimerized and autophosphorylated. The activated receptors further phosphorylate the receptor substrates to initiate intracellular kinase signaling cascades (1-3). It is evident, however, that there may be alternative signaling pathways for some growth factors involving their nuclear transport and signaling (for review see Ref. 4). In this context, subsequent to receptor-ligand internalization, growth factor ligands may translocate to the nucleus and directly function in mitogenic processes (5, 6). FGF-1 1 is one of two prototype members of the fibroblast growth factor family. It is a potent mitogen for many cell types and is involved in embryogenesis, angiogenesis, and neurite outgrowth (7,8). The mechanism by which FGF-1 transmits mitogenic signals is still not entirely clear. It has been shown, however, that a mutant FGF-1 with deletion in its nuclear localization sequence (NLS) Asn-Tyr-Lys-Lys-Pro-LysLeu (residues 21-27) failed to stimulate DNA synthesis and cell proliferation in vitro although it could still bind to the FGF receptor and induce intracellular receptor-mediated tyrosine phosphorylation and c-fos expression (5). The fact that FGF-1-(21-27) was able to direct ␤-galactosidase into the nucleus, as well as the evidence of nuclear localization of FGF-1 (9, 10), suggest that nuclear transport of FGF-1 following receptormediated internalization might be important for stimulating DNA synthesis by FGF-1 in vitro.To examine directly the functional role of the NLS in FGF-1-stimulated mitogenesis, we have delivered the peptide encompassing this sequence into living cells by using our recently developed cell-permeable peptide import method (CPPI) (see Ref. 11). We demonstrated in this report that cell membranepermeable peptides containing this NLS sequence can stimulate DNA synthesis in NIH 3T3 cells in a F...