Analysis of growth factor stimulated nucleocytoplasmic protein transport using two-dimensional gel electrophoresis.
The rapid activation of gene expression by growth factors frequently involves the nucleocytoplasmic redistribution of proteins which regulate the transcription of discrete sets of genes. In particular, it has become apparent that protein kinases and transcription factors including protein kinase A, mitogen activated protein kinase, protein kinase C, NF-kappa B and Stat91, can rapidly migrate from the cytosol to the nucleus in response to a wide variety of extracellular stimuli. Furthermore, it is assumed that these proteins represent important components of the signal transduction pathways which operate between the plasma membrane and the nucleus. Here two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to detect proteins which undergo rapid growth factor stimulated nucleocytoplasmic redistribution. Epidermal growth factor and insulin influenced the association of 15 individual protein spots with the nucleus. Estimates of the abundance of these proteins revealed that they represented approximately 1/1000 to 1/5000 of total nuclear protein. To permit identification by microsequencing, five of the protein spots have been recovered from multiple 2-D PAGE gels and concentrated by one-dimensional elution-concentration gel electrophoresis. The data obtained demonstrate that 2-D PAGE can be used to: (i) analyse putative regulators of growth factor activated gene transcription, and (ii) purify sufficient amounts of these proteins to facilitate identification by microsequence analysis.
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011869:Radioligand Assay; D015398:Signal Transduction / Q000187:drug effects*; D013268:Stimulation, Chemical; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic / Q000187:drug effects*
D006133:Growth Substances; D007328:Insulin; D009687:Nuclear Proteins; D013462:Sulfur Radioisotopes; D004815:Epidermal Growth Factor; D003513:Cycloheximide