Using the approaches of mass spectrometry-based phosphoproteomics and small interfering RNA (siRNA)-induced gene silencing to identify new insulin receptor signaling components in cultured adipocytes.
EMSL Project ID
5893
Abstract
Insulin is a hormone that plays key roles in the regulation of fuel (glucose, fatty acid and protein) metabolisms and gene expression in the human body. Impaired insulin signaling and actions lead to the development of diabetes, a devastating disease that is reaching epidemic proportions in the USA and worldwide. There is an urgent need for developing new therapeutic drug for diabetes. Understanding how insulin signal transduction leads to its metabolic functions is a critically important step for identifying potential therapeutic targets of insulin resistance and diabetes. However, it remains elusive that how insulin regulates cellular functions. In general, insulin binds to and activates insulin receptor tyrosine kinase on the cell surface of insulin sensitive tissues such as fat and skeletal muscles (for review see 1). It has been established that activation of the insulin receptor tyrosine kinase catalyses tyrosine phosphorylation of various intracellular protein substrates. The phosphorylated tyrosines in these substrates act as docking sites for signal molecules leading to the regulation of the function of multiple down-stream protein kinases such as phosphatidylinositol 3-kinase, protein kinase B (PKB), atypical protein kinase C (aPKC), glycogen synthase kinase, MAP kinase, P70 ribosomal S6 kinase and cytoplasmic tyrosine kinase Fyn. Activation of these kinases may result in phosphorylation of multiple intracellular proteins at tyrosine, serine and threonine residues since each of the kinase has various protein substrates, of which most are unknown. It is very likely that those phosphoproteins mediate the pleiotropic insulin response in the target cells. Identifying those phosphoproteins is critically important for understanding insulin signaling process and may provide potential therapeutic target(s) for diabetes.In an attempt to identify new components of insulin receptor signaling, we recently affinity purified phospho-antibodies that recognize serine/threonine (S/T) phosphorylation motifs corresponding to target sites of multiple protein serine/threonine kinases described above. Our preliminary data showed that the polyclonal antibodies are tremendous efficient for immunoprecipitation (IP) and immuno-detection of multiple phosphoproteins stimulated by insulin in cultured adipocytes (see Fig1). Using MALDI-TOF MS, we identified and verified two proteins from the insulin-stimulated cell lysate samples immunoprecipitated with the phospho-antibodies and dissolved in SDS-PAGE. However, most (>10) of the phosphoprotein bands detectable by immunoblotting with the phospho-antibodies have not been identified yet (Fig1). This could be due to the phophorylated proteins are not the major proteins presented in the relevant bands. To identify those phosphoproteins altogether, phosphopeptide-specific enrichment approaches may be required. First, total lysate from insulin-stimulated cells can be immunoprecipitated with the serine/threonine antibodies. Immunoprecipitated phosphoproteins can be tryptic digested and phospho-peptides enriched with an IMAC column. Finally, the phosphopeptides can be separated and identified with a LC-MS/MS system such as capillary reverse phase liquid chromatography (RPLC) coupled online with LCQ and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. LCQ MS can be used for identifying total proteins immunoprecipitated with the phospho-antibodies but it may also detect peptides from proteins that are not phosphorylated by insulin-stimulated kinases but associated with phosphoproteins and therefore may be pulled down by IP. From the point of understanding insulin signaling, it would be an ideal approach to use both LCQ and FTICR system together to identify the phosphorylation sites of phosphoproteins and their associated proteins. At 6th International Symposium on Mass Spectrometry in Molecular and Cellular Proteomics (August 24-28, 2003, San Francisco), Dr. Jeongkwon Kim and colleagues from Environmental Molecular Sciences Laboratory of PNNL presented an impressive postal demonstrating a successful application of FTICR MS system to analysis phosphorylation of chichen myelin basic protein (2). It is most likely that the technology developed by Drs. Jeongkwon Kim, Harold Udseth and colleagues would be helpful for identifying insulin-stimulated phosphoproteins. Here, I would like to propose the following steps to collaborate with Drs. Jeongkwon Kim and Harold Udseth at EMSL to identify and verify new components of insulin receptor signaling in adipocytes:
First, phosphoproteins from total cell lysate of insulin-stimulated mouse adipocytes will be immunoprecipitated with the phospho-antibodies. Multiple IP will be carried out in our lab at UMass and samples will be pooled together to generate enough phopshoproteins before sending to EMSL for tryptic digestion and IMAC purification. Second, phosphopeptides enriched with or without IMAC column will be analyzed by Dr. Kim and colleagues using RPLC-MS, and detected phosphopeptides, their phosphorylation sites, non-phosphopeptides and their corresponding mouse proteins will be listed. Finally, to prove the listed proteins are actually phosphorylated by a protein kinase in insulin receptor signaling process, three different biological assays will be carried out using our cell biology system at UMass Medical School. One of the assays is to compare the protein phosphorylation states under control and insulin-stimulated conditions. The experiments will be carried out by immunoprecipitating cell lysate with the phospho-antibodies followed by immunoblotting the IP sample with antibodies against the listed proteins. To approve that the phosphorylation site(s) detected by MS is actually responsible for insulin-stimulated protein phosphorylation in cultured cells, the phosphorylation site(s) in the listed protein will be mutated into alanine(s) and expressed in the cells. Then the effects of phosphorylation site-directed mutation on insulin-stimulated protein phosphorylation will be tested by IP and immunoblotting as described above and previously (3-5). In addition, we developed an efficient protocol for gene silencing in cultured adipocytes using short interfering RNA (siRNA) (6). With this powerful functional tool, we will design and transfect the adipocytes with siRNA to induce gene-specific silencing of the phosphoprotein(s) and then insulin?s actions on glucose transport, fatty acid transport and synthesis, protein synthesis in cultured adipocytes will be analyzed as described previously (6). It is most likely that any new phosphoprotein identified with significant cellular function(s) will allow us publish one or multiple papers together.
Project Details
Project type
Exploratory Research
Start Date
2003-12-01
End Date
2005-01-12
Status
Closed
Released Data Link
Team
Principal Investigator