The control of renal water excretion occurs in part by regulation of transcription in response to vasopressin in cells of the collecting duct. A systems biology-based approach to understanding transcriptional control in renal collecting duct cells depends on knowledge of what transcription factors and other regulatory proteins are present in the cells' nuclei. The goal of this article is to report comprehensive proteomic profiling of cellular fractions enriched in nuclear proteins from native inner medullary collecting duct (IMCD) cells of the rat. Multidimensional separation procedures and state-of-the art protein mass spectrometry produced 18 GB of spectral data that allowed the high-stringency identification of 5,048 proteins in nuclear pellet (NP) and nuclear extract (NE) fractions of biochemically isolated rat IMCD cells (URL: https://helixweb.nih.gov/ESBL/Database/IMCD_Nucleus/). The analysis identified 369 transcription factor proteins out of the 1,371 transcription factors coded by the rat genome. The analysis added 1,511 proteins to the recognized proteome of rat IMCD cells, now amounting to 8,290 unique proteins. Analysis of samples treated with the vasopressin analog dDAVP (1 nM for 30 min) or its vehicle revealed 99 proteins in the NP fraction and 88 proteins in the NE fraction with significant changes in spectral counts (Fisher exact test, P < 0.005). Among those altered by vasopressin were seven distinct histone proteins, all of which showed decreased abundance in the NP fraction, consistent with a possible effect of vasopressin to induce chromatin remodeling. The results provide a data resource for future studies of vasopressin-mediated transcriptional regulation in the renal collecting duct.
Phosphorylation of the aquaporin-2 (AQP2) water channel at four COOH-terminal serines plays a central role in the regulation of water permeability of the renal collecting duct. The level of phosphorylation at these sites is determined by a balance between phosphorylation by protein kinases and dephosphorylation by phosphatases. The phosphatases that dephosphorylate AQP2 have not been identified. Here, we use large-scale data integration techniques to identify serine-threonine phosphatases likely to interact with AQP2 in renal collecting duct principal cells. As a first step, we have created a comprehensive list of 38 S/T phosphatase catalytic subunits present in the mammalian genome. Then we used Bayes' theorem to integrate available information from large-scale data sets from proteomic and transcriptomic studies to rank the known S/T phosphatases with regard to the likelihood that they interact with AQP2 in renal collecting duct cells. To broaden the analysis, we have generated new proteomic data (LC-MS/MS) identifying 4538 distinct proteins including 22 S/T phosphatases in cytoplasmic fractions from native inner medullary collecting duct cells from rats. The official gene symbols corresponding to the top-ranked phosphatases (common names in parentheses) were: Ppp1cb (PP1-β), Ppm1g (PP2C), Ppp1ca (PP1-α), Ppp3ca (PP2-B or calcineurin), Ppp2ca (PP2A-α), Ppp1cc (PP1-γ), Ppp2cb (PP2A-β), Ppp6c (PP6C), and Ppp5c (PP5). This ranking correlates well with results of prior reductionist studies of ion and water channels in renal collecting duct cells.
Receptor tyrosine kinases (RTKs) are cell membrane proteins that provide cells with the ability to sense proteins in their environments. Many RTKs are essential to development and organ growth. Derangement of RTKs-by mutation or by overexpression-is central to several developmental and adult disorders including cancer, short stature, and vascular pathologies. The mechanism of action of RTKs is complex and is regulated by contextual components, including the existence of multiple competing ligands and receptors in many families, the intracellular location of the RTK, the dynamic and cell-specific coexpression of other RTKs, and the commonality of downstream signaling pathways. This means that both the state of the cell and the microenvironment outside the cell play a role, which makes sense given the pivotal location of RTKs as the nexus linking the extracellular milieu to intracellular signaling and modification of cell behavior. In this review, we describe these different contextual components through the lens of systems biology, in which both computational modeling and experimental "omics" approaches have been used to better understand RTK networks. The complexity of these networks is such that using these systems biology approaches is necessary to get a handle on the mechanisms of pathology and the design of therapeutics targeting RTKs. In particular, we describe in detail three concrete examples (involving ErbB3, VEGFR2, and AXL) that illustrate how systems approaches can reveal key mechanistic and therapeutic insights. This article is categorized under: Biological Mechanisms > Cell Signaling Models of Systems Properties and Processes > Mechanistic Models Translational, Genomic, and Systems Medicine > Therapeutic Methods K E Y W O R D S cell signaling, computational model, receptor tyrosine kinase, systems biology 1 | INTRODUCTION 1.1 | Receptor tyrosine kinases and their mechanisms of actionReceptor tyrosine kinases (RTKs) are key components linking the microenvironment outside of the cell to the signaling networks inside of the cell, and to consequent changes in cell decision-making and behavior (Figure 1). Just as integrins sense extracellular matrix proteins and mechanical strain, and just as cadherins make direct contact between adjacent cells, RTKs *These authors have contributed equally to this study.
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