The long term goal of this research is to discover and elucidate the series of molecular events that constitute the mechanism(s) of action of insulin. This proposal will exploit the insulin- stimulated phosphorylation of ribosomal protein S6 to dissect signaling mechanisms used by insulin. The properties of this response that make it logical-to use for this purpose are: 1) Its magnitude; insulin can increase the incorporation of labeled phosphate into S6 by 20-fold and the stoichiometry of phosphorylation from less than 1 up to 5 mol of phosphate per mol of S6. 2) Its stability; extracts from insulin-treated cells phosphorylate S6 up to 10-fold faster than extracts from untreated cells. Finally 3) the effect can be elicited by insulin in a cell lysate indicating a potential for reconstitution in vitro. Insulin-treated cells and tissues contain an insulin-stimulated protein kinase that can be purified in an activated form.
The specific aims of this proposal focus on the isolation and study of this insulin-stimulated ribosomal protein S6 kinase as follows: 1) purify it; 2) make antibodies to it; 3) clone it; 4) isolate its inactive form; 5) examine the mechanism of its regulation; and 6) examine its role in insulin-stimulated S6 phosphorylation in intact cells. Since the activated form of this S6 protein kinase can be isolated, study of this enzyme offers a means of identifying the intermediates in the pathway of its activation. Elucidation of the pathway of regulation of this enzyme by insulin will enable reconstruction of the series of steps that constitute one mechanism of action of the hormone.
| Thorne, Curtis A; Chen, Ina W; Sanman, Laura E et al. (2018) Enteroid Monolayers Reveal an Autonomous WNT and BMP Circuit Controlling Intestinal Epithelial Growth and Organization. Dev Cell 44:624-633.e4 |
| Guerra, Marcy L; Kalwat, Michael A; McGlynn, Kathleen et al. (2017) Sucralose activates an ERK1/2-ribosomal protein S6 signaling axis. FEBS Open Bio 7:174-186 |
| Kalwat, Michael A; Cobb, Melanie H (2017) Mechanisms of the amplifying pathway of insulin secretion in the ? cell. Pharmacol Ther 179:17-30 |
| Dyachok, Julia; Earnest, Svetlana; Iturraran, Erica N et al. (2016) Amino Acids Regulate mTORC1 by an Obligate Two-step Mechanism. J Biol Chem 291:22414-22426 |
| McReynolds, Andrea C; Karra, Aroon S; Li, Yan et al. (2016) Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding. Biochemistry 55:1909-17 |
| Jivan, Arif; Ranganathan, Aarati; Cobb, Melanie H (2010) Reconstitution of the nuclear transport of the MAP kinase ERK2. Methods Mol Biol 661:273-85 |
| Duan, Lingling; Cobb, Melanie H (2010) Calcineurin increases glucose activation of ERK1/2 by reversing negative feedback. Proc Natl Acad Sci U S A 107:22314-9 |
| Lidke, Diane S; Huang, Fang; Post, Janine N et al. (2010) ERK nuclear translocation is dimerization-independent but controlled by the rate of phosphorylation. J Biol Chem 285:3092-102 |
| Lawrence, Michael C; Shao, Chunli; McGlynn, Kathleen et al. (2009) Multiple chromatin-bound protein kinases assemble factors that regulate insulin gene transcription. Proc Natl Acad Sci U S A 106:22181-6 |
| Lawrence, Michael C; McGlynn, Kathleen; Shao, Chunli et al. (2008) Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in beta-cells. Proc Natl Acad Sci U S A 105:13315-20 |
Showing the most recent 10 out of 85 publications
