H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluidphase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on P2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes.The pleiotropic hormonal and growth-promoting effects of insulin are well known (1). These include insulin's stimulation of glucose, ion, and amino acid transport, effects on numerous enzymatic processes, and stimulation of protein synthesis and cellular proliferation. Also documented are insulin's positive or negative effects on the expression of a number of specific genes (2), several of which may be associated with cell growth and proliferation (3)(4)(5)(6)(7). The mechanisms by which insulin affects these cellular processes are relatively unknown. It is generally accepted that the first and obligatory step is insulin binding to its cell membrane receptor. The ensuing autophosphorylation of the receptor's ,B subunit and activation of the intrinsic tyrosine kinase activity (8) have been implicated, directly or indirectly, in some effects of insulin. However, recent evidence suggests that alternative second messenger-type mechanisms are involved in other insulin responses (9, 10). In addition, some insulin responses may be induced without insulin binding to the plasma membrane receptor. Miller (11) demonstrated that insulin microinjected into the cytoplasm of frog oocytes stimulated RNA and protein synthesis. Others showed that insulin added to isolated nuclei effected enzymatic (12) and transport processes (13,14).In a series of biochemical and ultrastructural studies we have reported that intact insulin accumulated in nuclei of 3T3-L1 adipocytes (15) and H35 hepatoma cells (13, 16) and associated with the nuclear matrix, which is involved with gene expression (17). Nuclear accumulation of insulin was temperature-and time-dependent but energy-independent. Although the nuclear accumulation of insulin resulted in part from receptor-mediated endocytosis, the plasma membrane receptor was not translocated to the nucleus (18). Interestingly, insulin accumulation in the nucleus continued to increase at media insulin concentrations in excess of those that saturated the plasma membrane receptors (16). This observation was, to our knowledge, the first indication that a non-receptor-mediated process also might be involved in insulin's nuclear translocation.In a recent study that investigated the nuclear translocation of insulin,...
Enhanced levels of disulfide-linked dimers of the neu oncogene product have been suggested to be associated with the transformed state [Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI: Nature 338:230-231, (1989)]. We, therefore, investigated the properties of the dimeric forms of p185HER-2/neu from the human breast carcinoma cell line, SK-BR-3. We found disulfide-linked dimers as well as noncovalently associated dimers that were detected by cross-linking with bis(sulfosuccinimidyl) suberate (BS3). However, the disulfide-linked dimers did not exist in intact cells, since they were eliminated when the cells were lysed in the presence of the alkylating agent, sodium iodoacetate. Moreover, the disulfide-linked dimeric molecules were not the activated form of p185HER-2 since they incorporated about the same level of phosphate in an in vitro kinase reaction as the monomeric molecules. In contrast, the noncovalent dimers appeared to be present on the surface of intact cells and were phosphorylated at levels at least tenfold higher than monomers in an in vitro kinase reaction.
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