Involvement of MAP kinase in insulin signalling revealed by non-invasive imaging of luciferase gene expression in single living cells.
BACKGROUND
Studies of the mechanisms by which signals are transmitted from receptor tyrosine kinases would be facilitated by a way of monitoring events at the single-cell level. We have explored how luciferase imaging can be used to examine the role of specific signalling pathways in insulin-stimulated gene expression. The analysis of luciferase expression in single cells has previously been hampered by the insensitivity of existing methodologies and the lack of a way of monitoring quantitatively, and independently, more than one promoter within the same cell. We have developed a technique for examining the dynamics of insulin-stimulated AP-1-dependent transcription in single living cells, and have explored the signalling pathway involved.


RESULTS
Luciferase and aequorin gene expression were examined in single living cells with a high-sensitivity photon-counting camera. The technique involved the comicroinjection of luciferase- and aequorin-based reporter plasmids directly into the cell nucleus, and the subsequent analysis of luminescence in the presence of luciferin and coelenterazine, respectively. The method is quantitative and allows insulin-stimulated gene expression to be monitored in real time. We found that insulin promoted a substantial increase in the expression of a luciferase gene under the control of the AP-1-binding site from the collagenase gene promoter. Aequorin expression, under the control of a cytomegalovirus promoter, was unaffected by insulin. The effect of insulin on luciferase expression was specifically blocked by overexpression of either the mitogen-activated protein (MAP) kinase phosphatase CL100, or the dominant-negative mutant MAP kinase kinase, MEKS217/221A.


CONCLUSIONS
Microinjection coupled with luciferase imaging allows hormone-regulated gene expression from relatively weak promoters to be monitored in single living cells. We have used this method to demonstrate that MAP kinase plays a central role in the ability of insulin to stimulate AP-1-dependent gene transcription.
Rutter
G A
GA
Department of Biochemistry, School of Medical Sciences, University of Bristol, UK.
White
M R
MR
Tavaré
J M
JM
D000331:Aequorin / Q000235:genetics; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D017364:Collagenases / Q000235:genetics; D006224:Cricetinae; D054638:Dual Specificity Phosphatase 1; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007091:Image Processing, Computer-Assisted; D017874:Immediate-Early Proteins / Q000235:genetics*; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics*; D008845:Microinjections; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D007328:Insulin; D018808:Transcription Factor AP-1; D000331:Aequorin; D008156:Luciferases; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; D017364:Collagenases