In response to insulin, protein-tyrosine phosphatase 1B (PTPase 1B) dephosphorylates 95-and 160 -180-kDa tyrosine phosphorylated (PY) proteins (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810 -19816 Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B complexes with the autophosphorylated insulin receptor in intact cells, either directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within both the receptor and PTPase 1B.Insulin is a potent metabolic and growth-promoting hormone that has pleiotropic effects at the level of the cell and within the intact organism. Insulin acts on cells to stimulate glucose, protein, and lipid metabolism, as well as RNA and DNA synthesis, by modifying the activity of a variety of enzymes and transport processes (1). As a first step in initiating these responses, insulin binds to its plasma membrane receptor. The insulin receptor is a heterotetrameric protein consisting of two ␣ and two ␤ subunits linked by disulfide bonds to form a ␤-␣-␣-␤ structure. After insulin binding, the insulin receptor undergoes autophosphorylation on tyrosine residues. Autophosphorylation increases the tyrosine kinase activity of the insulin receptor, which in turn phosphorylates one or more cellular substrates, leading to a cascade of secondary phosphorylation and dephosphorylation reactions (2).As the molecular mechanism of insulin action is defined with increasing clarity, so too is our appreciation of the central role played by protein tyrosine phosphorylation. Regulated tyrosine phosphorylation represents a balance of protein-tyrosine kinase (PTKase) 1 and protein-tyrosine phosphatase (PTPase) activities. To date, most attempts to assess the role of proteintyrosine phosphorylation in insulin signal transduction have focused on the action of kinases and thus furnish an incomplete picture of this dynamic process. PTPases can be used as probes to test the role of protein tyrosine phosphorylation, complementing studies performed on the PTKases.Extensive progress in the identification and characterization of PTPases has been made in recent years (3), partially as a result of our appreciation of the PTKases (4). PTPase 1B was the first PTPase to be isolated in homogeneous form and sequenced (5, 6). PTPase 1B possesses a catalytic domain characterized by the 11-amino acid sequence motif, (I/V)HCX-AGXXR(S/T)G. This motif contains cysteine (Cys 215 ) and arginine (Arg 221 ) residues critical for the catalytic activity of the enzyme (7-9). The cDNA sequences for human (10, 11) and rat (8) PTPase 1B predict a protein of 50 kDa with 435 and 432 amino acids, respectively. The conserved phosphatase domain of PTPase 1B is contained within the domain spanning residues 30 to 278. The COOH-terminal noncatalytic extension of the protein serves a regulatory function. The COOH-terminal 35 residues target the ...

Binding of extracellular ligands to epidermal growth factor receptors (EGFR) activate signal transduction pathways associated with cell proliferation, and these events are inhibited by monoclonal antibodies against EGFR. Since efficient DNA repair in actively growing cells may require growth factor signaling, it was of interest to explore any linkage between EGFR-mediated signaling and DNA-dependent protein kinase (DNA-PK), an enzyme believed to be involved in repairing double strand breaks and V(D)J recombination. We report that anti-EGFR monoclonal antibodies (mAbs), and not EGFR ligands, trigger a specific early physical interaction between EGFR and a 350-kDa catalytic subunit of DNA or its regulatory heterodimeric complex Ku70/80, in a variety of cell types, both in vivo and in vitro. Inhibition of EGFR signaling by anti-EGFR mAb was accompanied by a reduction in the levels of the DNA-PK and its activity in the nuclear fraction. Confocal imaging revealed that a substantial amount of DNA-PK was co-localized with EGFR in anti-EGFR mAb-treated cells. Anti-EGFR mAb-induced physical interaction between EGFR and DNA-PK or Ku70/80 was dependent on the presence of EGFR, but not on the levels of EGFR. The EGFR associated with DNA-PK or Ku70/80 retains its intrinsic kinase activity. Our findings demonstrate the existence of a novel cellular pathway in mammalian cells that involves physical interactions between EGFR and DNA-PK or Ku70/80 in response to inhibition of EGFR signaling. Our present observations suggest a possible role of EGFR signaling in maintenance of the nuclear levels of DNA-PK, and interference in EGFR signaling may possibly result in the impairment of DNA repair activity in the nuclei in anti-EGFR mAbtreated cells.

Summary The retinoblastoma (RB)/p16INK4a pathway regulates senescence of human melanocytes in culture and oncogeneinduced senescence of melanocytic nevi in vivo . This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16 INK4a -positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/ dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracyclinebased inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega-complexes, formation of heterochromatin protein 1β β β β (HP1β β β β )/SUV39H1 foci, methylation of H3-K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent-associated β β β β -galactosidase marker. Notably, formation of RB/HP1β β β β foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1β β β β /RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events.