Elsevier

Methods in Enzymology

Volume 568, 2016, Pages 113-138
Methods in Enzymology

Chapter Five - Assays for Posttranslational Modifications of Intermediate Filament Proteins

https://doi.org/10.1016/bs.mie.2015.09.005Get rights and content

Abstract

Intermediate filament (IF) proteins are known to be regulated by a number of posttranslational modifications (PTMs). Phosphorylation is the best-studied IF PTM, whereas ubiquitination, sumoylation, acetylation, glycosylation, ADP-ribosylation, farnesylation, and transamidation are less understood in functional terms but are known to regulate specific IFs under various contexts. The number and diversity of IF PTMs is certain to grow along with rapid advances in proteomic technologies. Therefore, the need for a greater understanding of the implications of PTMs to the structure, organization, and function of the IF cytoskeleton has become more apparent with the increased availability of data from global profiling studies of normal and diseased specimens. This chapter will provide information on established methods for the isolation and monitoring of IF PTMs along with the key reagents that are necessary to carry out these experiments.

Introduction

Intermediate filament (IF) proteins are important for the maintenance of cellular function in the basal state, and are particularly important under stress and in disease states (Davidson and Lammerding, 2014, Gruenbaum and Aebi, 2014, Homberg and Magin, 2014, Omary, 2009, Toivola et al., 2015). IFs are major structural components of the cell cytoskeleton, but through their dynamic behavior and under varying cellular conditions, they have also been demonstrated to impact virtually every aspect of cellular function, including gene transcription, signaling pathways, and cellular survival (Chung et al., 2013, Herrmann et al., 2009, Toivola et al., 2010). The assembly and disassembly dynamics of IF proteins, as well as their associations with other cellular components are regulated by various posttranslational modifications (PTMs), summarized in Table 1, and a myriad of enzymes that carry out specific PTM on/off reactions (Hyder et al., 2008, Omary et al., 2006, Snider and Omary, 2014).

The extent of functional understanding regarding the role of each PTM on IF protein function is highly dependent on the availability of tools to study the particular PTM of interest. For example, phosphorylation (Roux & Thibault, 2013) and ubiquitination (Sylvestersen, Young, & Nielsen, 2013) can be analyzed using mass spectrometry with relative ease, whereas sumoylation (Gareau & Lima, 2010), which has relatively low stoichiometry and is not easily analyzed by mass spectrometric means, is more difficult to probe. Therefore, the system-level PTM data currently available are skewed to highlight those PTMs that can be readily tracked using proteomic platforms (Choudhary and Mann, 2010, Hennrich and Gavin, 2015). The combination of global proteomic data with PTM databases that catalog experimentally determined and site-specific modifications, or that use computational approaches to predict and quantify PTMs (Table 2), has resulted in a wealth of information on modified residues on IF proteins. However, most of these modifications await functional assignment. For most IF protein PTMs, the use of molecular approaches (e.g., site-directed mutagenesis of modification sites), biochemical tools (pan- or site-specific PTM antibodies), chemical probes (inhibitors or activators of PTM enzymes), and transgenic mouse models, in combination with enrichment of the IF protein fraction from cells and tissues, has yielded useful insight into some of the functional roles of PTMs, although much more remains to be learned. The relative insolubility of IF proteins (particularly epidermal keratins) in nondenaturing detergent-containing buffers can be an impediment to the study of PTMs, although these limitations can be surmounted, as was shown for the type I keratin K17 (Pan, Kane, Van Eyk, & Coulombe, 2011).

PTMs participate in complex cross-talk mechanisms to regulate IF function. The balance of various modified forms of IF proteins is dictated by cellular conditions, such as mitosis, cell migration, stress, and apoptosis. The key to resolving the information encoded by IF PTMs is to determine which PTM signatures are prevalent under a given condition and how altering the stoichiometry of IF PTMs alters IF function, distribution, interactions, and ultimately, cellular fate.

Using the database PhosphoSitePlus (Hornbeck et al., 2015), we conducted a search for PTMs on human keratin 8 (K8) that have been reported by at least one low-throughput study, or those that appear in at least five high-throughput studies/records (Table 3). In this case, low-throughput refers to data generated via amino acid sequencing, site-directed mutagenesis, or the use of specific antibodies, whereas high throughput refers to studies using unbiased discovery-mode mass spectrometry. This example analysis revealed that 16% (76/483) of residues on K8 are modified by either phosphorylation, acetylation, ubiquitination, sumoylation, or methylation. The majority of the PTM sites on K8 (47/76) are modified by phosphorylation (pSer > pTyr > pThr). It can also be appreciated that most of the acetylation sites are also targets for ubiquitination, and one residue (Lys-285) appears to be capable of undergoing sumoylation, acetylation, or ubiquitination. These observations suggest that, depending on the cellular conditions, the various PTMs may directly compete for a given site. In addition to direct competition, there is experimental evidence for functional cross-talk between the different PTMs, such as between phosphorylation and ubiquitination, sumoylation, and acetylation (Snider & Omary, 2014).

Regulation of IF proteins by PTMs occurs under specific cellular conditions, which can be modeled using chemical and pharmacological agents. An abbreviated list of representative reagents is shown in Table 4. Hyperphosphorylation of IF proteins can be induced by treatment with phosphatase inhibitors, such as okadaic acid or microcystin-LR. Phosphorylation, in turn, can promote IF protein sumoylation, so these two modifications are generally observed together, particularly under conditions of oxidative stress and apoptosis (Snider, Weerasinghe, Iniguez-Lluhi, Herrmann, & Omary, 2011). The latter can be modeled in cells using hydrogen peroxide and anisomycin, respectively, among numerous other reagents that can induce similar responses. Increased acetylation of K8 in epithelial cells is dose–responsive to glucose levels in vitro and in vivo, and can be induced more robustly if the cells undergo transient glucose starvation prior to restimulation (Snider, Leonard, et al., 2013). Increased O-glycosylation of K18 in vitro and in vivo is observed in response to PUGNAc (Ku, Toivola, Strnad, & Omary, 2010), an inhibitor of the enzyme that removes O-linked N-acetylglucosamine (O-GlcNAc) groups (O-GlcNAcase). Since many IF proteins (Rogel, Jaitovich, & Ridge, 2010) are turned over by ubiquitination and proteasomal degradation, treatment with the proteasome inhibitor MG-132 promotes the accumulation of poly-ubiquitinated IF proteins. The sections that follow describe the general methods for isolation of IF proteins from cells and tissues, followed by a summary of specific tools and approaches to study more common PTMs on IF proteins.

Section snippets

Materials and Reagents for Isolation of IF Proteins

Source of IF protein:

  1. Human or animal tissue

  2. Primary cells (e.g., freshly isolated hepatocytes)

  3. Cultured cells expressing endogenous IF proteins

  4. Cultured cells overexpressing IF protein(s) of interest

Antibodies and immunoprecipitation beads:
  1. Anti-IF protein antibodies

  2. Anti-PTM antibodies (pan- or site-specific)

  3. IgG isotype control

  4. Dynabeads magnetic beads (or similar)

Buffers:
  1. TXB: Triton-X Buffer

    • 1% Triton-X-100

    • 5 mM EDTA

    • Bring up volume in PBS, pH 7.4

  2. TXB + PPI: Triton-X Buffer with Protease and Phosphatase Inhibitors

    Immediately before use, add protease and phosphatase

Methods for Monitoring Specific PTMs on IF Proteins

The sections below describe the functional significance and the available tools and methods to study several IF protein PTMs: phosphorylation, sumoylation, acetylation, glycosylation, and transamidation. For specific methods for the analysis of other PTMs, such as farnesylation (Hannoush & Sun, 2010), ubiquitination (Lill & Wertz, 2014), and ADP-ribosylation (Moyle & Muir, 2010), we direct the readers to general protocols and previous applications of these modifications in the context of IF

Conclusions

There are several hundred known PTMs across the proteome, and we are just beginning to understand the information that they encode in order to promote adaptable functions of proteins under specific physiological and pathophysiological conditions. IF proteins are known to be regulated by a number of PTMs, but with the improvement in detection methods we will be able to uncover more details about the known IF PTMs as well as identify novel IF-targeting PTMs. Generation of new site-specific

Acknowledgments

The authors' work is supported by US National Institutes of Health (NIH) grants DK47918 and DK52951, and the Department of Veterans Affairs (M.B.O.); NIH grants DK093776 and DK102450 (N.T.S.); and NIH institutional grant DK34933 to the University of Michigan. M.B.O. expresses gratitude to his laboratory coworkers who have made significant contributions to our understanding of IF PTMs including Carrie Riopel, Chih-Fong Chou, Diana Toivola, Guo-Zhong Tao, Jian Liao, Li Feng, Lori Lowthert, Masaru

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