Changes In Phosphorylation State Of Proteins Research Articles

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Quantitative phosphoproteomic analyses of the inferior parietal lobule from three different pathological stages of Alzheimer's disease.

Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is clinically characterized by progressive neuronal loss resulting in loss of memory and dementia. AD is histopathologically characterized by the extensive distribution of senile plaques and neurofibrillary tangles, and synapse loss. Amnestic mild cognitive impairment (MCI) is generally accepted to be an early stage of AD. MCI subjects have pathology and symptoms that fall on the scale intermediately between 'normal' cognition with little or no pathology and AD. A rare number of individuals, who exhibit normal cognition on psychometric tests but whose brains show widespread postmortem AD pathology, are classified as 'asymptomatic' or 'preclinical' AD (PCAD). In this study, we evaluated changes in protein phosphorylation states in the inferior parietal lobule of subjects with AD, MCI, PCAD, and control brain using a 2-D PAGE proteomics approach in conjunction with Pro-Q Diamond phosphoprotein staining. Statistically significant changes in phosphorylation levels were found in 19 proteins involved in energy metabolism, neuronal plasticity, signal transduction, and oxidative stress response. Changes in the disease state phosphoproteome may provide insights into underlying mechanisms for the preservation of memory with expansive AD pathology in PCAD and the progressive memory loss in amnestic MCI that escalates to the dementia and the characteristic pathology of AD brain.

Abstract 1608: Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells

Abstract Tyrosine kinase inhibitors (TKIs) against c-MET and EGFR have positive therapeutic effects in a subset of Non-Small Cell Lung Cancer (NSCLC) patients; however, their clinical efficacies are limited due to the development of TKI resistance. To determine mechanisms of TKI resistance, two NSCLC model cell lines H2170 and H358 were made resistant to the c-Met/EGFR TKIs SU11274, erlotinib or a combination of both by exposing the cells to progressively increasing concentrations of inhibitor. Initial characterization of these cell lines using phospho-specific antibodies showed that phospho-c-Met was found to be downregulated 4- and 1.5- fold in SU11274-resistant (SR) H2170 and SR H358 cells, respectively. Phospho-EGFR was found to be constitutively autophosphorylated (upregulated 19-fold) in erlotinib-resistant (ER) H2170 cells, but downregulated 6-fold in ER H358 cells. Interestingly, c-Met/EGFR downstream signaling proteins p-mTOR, p-p70S6K and p-ERK were found to be 2-4-fold upregulated, 2-fold upregulated and 2-5-fold upregulated, respectively, in ER H2170 and H358 cells. Phospho-p70S6K was also found to be upregulated 20-fold in SR H2170 cells. These results indicate that alternative signaling pathways may confer TKI resistance in these cell lines. To identify which signaling pathways may be responsible for TKI resistance, H358 parental and resistant cell lines were analyzed by mass spectrometry (MS). Differences in protein expression in cell lines treated with and without EGF and/or erlotinib were measured using higher multiplexing Thermo Scientific™ Tandem Mass Tag™ (TMT™) reagents. More than 1500 protein groups were identified and quantified using a Thermo Scientific™ Orbitrap™ Fusion™ platform with HCD-MS2 and MS3 fragmentation methods. Protein expression in ER H358 cells showed a marked increase in expression of DNA replication proteins, transcription factors, apoptosis inhibitors and some Ras-associated signal transduction pathways. To further investigate the mechanism of TKI resistance, phospho-peptide enrichment was also used in combination with TMT quantitation to profile changes in protein phosphorylation state. Overall, the combination of phospho-peptide enrichment coupled with higher multiplexing TMT and high resolution MS analysis enabled deeper global profiling of TKI resistance signaling pathways compared to previous studies without enrichment. Citation Format: Ryan D. Bomgarden, Ryan Jacobs, Jason Fong, David Moravec, Gregory M. Botting, Michael Blank, Rosa I. Viner, John C. Rogers, Neelu Puri. Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1608. doi:10.1158/1538-7445.AM2014-1608

Abstract 1248: The use of nanoimmunoassay technology to predict response to insulin-like growth factor-1 receptor inhibition in head and neck squamous carcinoma

Abstract Background: Signaling from the insulin-like growth factor-1 receptor (IGF1R) can play a role in resistance to anti-cancer therapy in head and neck squamous cell carcinoma (HNSCC). Thus, targeted inhibition of the IGF1R holds substantial therapeutic potential. However, while several small molecule tyrosine kinase inhibitors (TKIs) of the IGF1R are in clinical trials, there is no definitive biomarker that predicts tumor responsiveness to anti-IGF1R therapy. It is likely that such a predictive biomarker would be a component of the most prominent downstream signaling cascades from the IGF1R, such as the MEK/ERK pathway or the PI3K/AKT pathway. Aims: (1) Identify a short-term predictor of long-term growth inhibition when the IGF1R-TKI OSI-906 is used alone or combined with an EGFR-TKI or cisplatin in HNSCC cell lines. (2) Validate a nanoscale method to quantitate ERK inhibition as a marker of growth inhibition and caspase-3 cleavage as a marker of apoptosis which can ultimately be used in preclinical and clinical studies. Methods: A novel and highly sensitive nanofluidic immunoassay (NIA) that is highly efficient at detecting changes in protein phosphorylation states has recently been developed. This emerging technology uses isoelectric focusing to characterize proteins in extremely small samples such as fine needle aspirate (FNA) biopsies. The utility of NIA was initially tested on Cal27 cells in vitro and studies were subsequently extended to Cal27 xenograft tumors in nude mice. Results: Using the NIA approach, basal and treated levels of unphosphorylated, monophosphorylated, and disphosphorylated ERK1 and ERK2 could be resolved using 40 ng of Cal27 cell lysate. Treatment with 1uM of OSI-906 or gefitinib resulted in 90% reduction in phospho-ERK species; the combination of both drugs completely eliminated ERK phosphorylation. Using the same approach, both drugs were noted to induce caspase-3 cleavage, which was greatest with combination treatment. These data correlate with reduced cell number and increased apoptosis at 24-72 h using standard assays. Preliminary studies using Cal27 xenografts in nude mice demonstrate the ability to detect ERK and caspase-3 in FNA biopsies of the tumors. Conclusion: Using NIA technology, ERK phosphorylation and/or caspase-3 cleavage may have utility as early markers of response when HNSCC cells or tumors are treated with TKIs or other drugs. This approach allows for micro- or nanoscale studies in vitro and simplifies assessment of very small tissue samples. As such, it has the potential to be used as a tool to determine the response of TKIs in patients at the molecular level without waiting for a clinical response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1248. doi:1538-7445.AM2012-1248

Profiling Protein Tyrosine Phosphorylation: A Quantitative 45-Plex Peptide-Based Immunoassay

Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor-stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications.

Data-driven modeling of cellular stimulation, signaling and output response in RAW 264.7 cells

BackgroundUnderstanding the relative importance of signaling pathway components which regulate a specific cellular response is a major focus of current efforts in biology. This interest, along with the inherit complexity of these systems, is driving the development of approaches capable of providing both quantitative predictions as well as guiding the design of future experiments. Of particular interest is the establishment of methods for the analysis of cellular-level input-output signaling relationships that have been characterized over time.ResultsWork by the Alliance for Cellular Signaling (AfCS) has provided an extensive profile of ligand-induced changes in protein phosphorylation state and cytokine output response in macrophage-like RAW 264.7 cells. Using model averaging with partial least squares (PLS) or principal components regression (PCR), we compared multivariate models quantitatively predicting cytokine release and identifying key regulatory components of the underlying signaling pathways. We paid particular attention to the effect of metrics extracted from the experimentally derived signaling time courses so as to determine whether the inclusion of such temporal information improved model predictions. Results indicate that we were able to determine the key biological predictors responsible for generating a specific cytokine response, with model R2 values ranging from 0.48 to 0.93. Furthermore, for this data set, the use of time metrics was found to be of mixed value, with increased and/or more appropriate sampling likely being required to improve predictive performance.ConclusionThe use of multivariate approaches and model averaging provides a valuable predictive framework for quantitative studies of these complex biological processes. Results of this work also point to several issues for consideration in the design of similar large-scale interrogations.