Subcellular fractionation reveals proteins involved in insulin signaling
phosphorylated themselves but bound to phosphotyrosine proteins. The researchers did not expect to The insulin signaling pathway is as identify CASK with the subcellular frac sociated with many cellular process tionation method because the pro es. When the regulation of this pathway tein is not known to play a role in insu lin-mediated processes. It is involved in goes awry, diseases and conditions, including diabetes, atherosclerosis, protein targeting, vesicle transport, and and obesity, can develop. To under transcriptional regulation in neuronal stand how insulin causes these effects, cells, but its function in other cell types Zhengjun Chen, Rong Zeng, and cois unknown, Chen says. Although west ern blots of anti-CASK immunoprecip workers at the Chinese Academy of Sci ences performed a proteomics study itates show that CASK is not phospho in which they looked for phosphotyro rylated on tyrosine residues, the protein sine-modified proteins in co-purifies with two phos various subcellular frac photyrosine proteins. DAPI CASK Merge To investigate whether tions. They chose this ap proach over analyzing total CASK is really involved in cell lysates because acti insulin signaling, the re vated insulin receptor (IR) searchers determined the Insulin (–) triggers a signaling cascade localization and function that involves tyrosine phos of the protein when insulin phorylation on several pro was present. Western blot teins located in different analysis showed that in the cellular compartments. The presence of insulin, CASK Insulin (+) work is described in this is levels decrease in cytoplas sue of JPR (pp 846–855). mic and nuclear fractions According to Chen, tradi but increase in membrane tional methods of focusing fractions. In immunoflu on one or a few signaling orescence studies, CASK On the move. Insulin treatment causes CASK to leave the nucleus, which proteins at a time are lim staining was not observed is stained with 4′,6-diamidino-2-phenylindole (DAPI). iting, so he decided to col in the nucleus when insulin laborate with Zeng and her was added to the cells. Nu clear CASK has been reported to co-ac proteomics experts. Previous attempts ment, as expected. Surprisingly, the to characterize the insulin signaling pattern of proteins in the cytoplasmic tivate the transcription of genes that have a T-element sequence, and the pathway typically involved an enrich fraction was markedly different from ment of phosphotyrosine proteins from those in the other fractions and in the transcription of a reporter gene is en total-cell lysates by affinity purification total-cell lysate. “This result can tell us hanced when the protein is co-trans fected with a T-element-containing or immobilized metal affinity chro that the subcellular fractionation is an matography (commonly called IMAC) absolutely necessary step to compre vector into Rat1/hIR cells. However, in methods. Chen says that the disadvan hensively isolate phosphotyrosine pro sulin treatment inhibited transcription tage to this type of study design is that teins and their complexes and to reduce of the reporter gene. Together, these re “non-tyrosine-phosphorylated proteins contamination of the purification,” sults suggest that CASK is involved in can be lost during sample preparation says Chen. “In addition, information the insulin signaling pathway, says and phosphopeptide purification.” In about identified proteins from different Chen. addition, some highly phosphorylat fractions may give us lots of hints con In the near future, Chen plans to val ed proteins can titrate out phosphoty cerning the localizations of the identi idate some of the other proteins identi rosine antibodies used for purification. fied proteins.” fied in the study and to perform quan Information about where particular Phosphotyrosine proteins and their titative analyses with the stable isotope proteins are located within a cell is also binding partners were analyzed by LC/ labeling with amino acids in cell cul lost when total lysates are used. MS/MS. The researchers identified 1100 ture (commonly known as SILAC) meth To overcome these challenges, the re proteins. In addition, 35 phosphotyro od. Chen says that the subcellular frac searchers fractionated Rat1/hIR cells sine sites were pinpointed on 31 unique tionation step could be used in other that stably overexpress the human form peptides. Some of the phosphorylations studies to uncover additional unexpect of IR. Cells that overexpress IR “made it occurred on novel sites, and some of the ed links between signaling pathways. possible for us to detect many weak sig identified phosphotyrosine proteins This information could help research nals that exist in normal native cells but were not previously known to be in ers to better understand signaling net works and to design more effective are otherwise undetectable due to the volved in insulin signaling. Other pro teins, such as calcium/calmodulin-de very low expression level of IR in most drugs. cells,” explains Chen. Insulin-treated pendent serine kinase (CASK), were not —Katie Cottingham ZHENGJUN CHEN
and control cells were separated into nuclear and non-nuclear fractions with hypotonic buffer. The non-nuclear part was then separated into cytoplasmic and membrane fractions by centrifuga tion. Immunoprecipitations with antiphosphotyrosine antibody were then performed on each fraction. The researchers analyzed the phos photyrosine protein content of each fraction and of the total-cell lysate by western blotting with anti-phosphoty rosine antibody. Tyrosine phosphory lation was enhanced by insulin treat
Journal of Proteome Research • Vol. 5, No. 4, 2006 739
