PTM: Phosphorylation

• INTRODUCTION

  Phosphorylation is a fundamental post-translational modification that plays a crucial role in regulating the function and activity of proteins. It involves the addition of a phosphate group to specific amino acid residues, typically serine, threonine, or tyrosine, in a protein molecule. This modification is dynamically regulated by protein kinases, which catalyze the transfer of the phosphate group from ATP to the target protein, and protein phosphatases, which remove the phosphate group. Understanding the intricacies of protein phosphorylation and its regulatory roles provides insights into the complexity of cellular signaling networks and opens avenues for targeted therapeutic interventions. By deciphering the specific phosphorylation events and their functional consequences, researchers can unravel the underlying mechanisms governing cellular processes and potentially develop strategies to modulate protein function for therapeutic purposes.

  Phosphorytion occurs in all organisms and very little has been studied on the role of phosphorylation of MDH in any organism. Thus this area is wide open for interesting research for any of the MDH clones available to MCC faculty. This project description will focus on both the cytosolic and mitochondrial isoforms of human MDH. Both isoforms have many phosphorylation sites, but more needs to be studied for mammalian MDH phosphorylation. Interestingly GOT, PEPCK and ME1 all enzymes that share substrates with MDH are also phosphorylated and methylated.  The mitochondrial GOT-MDH and CS-MDH may very well be regulated not by phosphorylation but acetylation.  There are, surprisingly very few publications of cytosolic MDH interactions with other proteins. 

  There are several examples of phosphorylation and regulation of dehydrogenases providing a mechanism for cells to modulate their activity in response to various signals and metabolic conditions.. One example is pyruvate dehydrogenase (PDH), a key enzyme involved in cellular energy metabolism. PDH catalyzes the conversion of pyruvate to acetyl-CoA, which enters the citric acid cycle. PDH is regulated by phosphorylation through the action of pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). Phosphorylation of PDH by PDK inhibits its activity, reducing the conversion of pyruvate to acetyl-CoA and thereby regulating the entry of pyruvate into the citric acid cycle. Conversely, dephosphorylation of PDH by PDP activates the enzyme. Another example is isocitrate dehydrogenase (IDH), a critical enzyme in the citric acid cycle. In certain cellular contexts, IDH can be regulated by phosphorylation. Phosphorylation of IDH can modulate its catalytic activity and affect the balance of metabolites in cellular metabolism.

  Lactate dehydrogenase (LDH) is closely related to MDH and can undergo phosphorylation, although the regulatory effects and functional consequences of this modification are still being studied and understood. The phosphorylation of LDH can potentially regulate its activity, stability, subcellular localization, or interactions with other molecules. Phosphorylation of LDH may be mediated by protein kinases, including protein kinase A (PKA) and protein kinase C (PKC), among others. The specific kinases involved and the functional consequences of LDH phosphorylation can vary depending on the cellular context and signaling pathways involved. While the exact role of LDH phosphorylation is still being elucidated, some studies suggest that phosphorylation may influence LDH activity and its association with other proteins in cellular processes related to metabolism, signaling, and cellular stress response.

  Like LDH, phosphorylation of MDH may potentially regulate its activity or interaction with other molecules. While there is limited research specifically focusing on MDH phosphorylation, some studies have reported phosphorylation events on MDH in certain contexts. For example, in plants, phosphorylation of MDH has been observed under various stress conditions, including drought, salt, and oxidative stress. The specific kinases involved and the functional consequences of MDH phosphorylation in these stress responses are still being investigated. Additionally, MDH can be phosphorylated indirectly as part of larger protein complexes or signaling cascades. For example, in certain cellular processes, such as gluconeogenesis, the phosphorylation status of enzymes upstream or downstream of MDH can indirectly influence its activity or regulation.

  The exact mechanisms, regulatory effects, and functional consequences of MDH phosphorylation are still areas of ongoing research, and more studies are needed to fully elucidate the significance of MDH phosphorylation in cellular metabolism and physiology.

• PROJECT IDEAS

  Phosphomimics refer to site directed mutations or substitutions in the coding region of a protein that mimic the effects of phosphorylation. Phosphorylation is a common post-translational modification involving the addition of a phosphate group to specific amino acid residues, typically serine, threonine, or tyrosine. Phosphomimics are designed to mimic the presence of a phosphate group on the protein without actually undergoing phosphorylation. By introducing specific amino acid substitutions, usually aspartic acid (D) or glutamic acid (E), the negative charge and bulkiness of the phosphate group can be mimicked. These substitutions can alter the charge, conformation, and interactions of the protein, leading to functional changes that may resemble the effects of phosphorylation.

  Phosphomimics are used in experimental studies to investigate the functional consequences of protein phosphorylation or to gain insights into phosphorylation-mediated signaling pathways. They can help researchers understand the regulatory role of phosphorylation on specific proteins or dissect the molecular mechanisms underlying phosphorylation-dependent processes.

  It is important to note that while phosphomimics can provide valuable insights, they do not fully replicate the complex and dynamic nature of phosphorylation. Phosphorylation is a tightly regulated process with precise spatial and temporal control, involving specific kinases and phosphatases. Phosphomimics serve as useful tools in research but should be interpreted cautiously, as they cannot fully capture the intricacies of native phosphorylation events.

  Creating a phosphomimic for phosphorylated tyrosine residues is more challenging compared to mimicking phosphorylation on serine or threonine residues. This is primarily because the phosphate group on tyrosine phosphorylation carries a distinct chemical and electrostatic character. Unlike serine and threonine phosphorylation, which introduces a negative charge via a phosphate group, tyrosine phosphorylation introduces a bulkier and aromatic phosphotyrosine residue. Consequently, mimicking the effects of tyrosine phosphorylation through simple amino acid substitutions is more difficult.

  Evidence of Phosphorylation: Several databases include sites of MDH phosphorylation - however there is very little published examining the phosphorylation or effect on MDH structure or function. A dive into several mass spec databases will show several sites of both cytosolic and mitochondrial human MDH. In yeast, one phosphorylation site on the N terminus of MDH changes the half-life of the protein. In a phosphoproteomic study conducted on the mitochondria of Saccharomyces cerevisiae (yeast), potential phosphorylation sites on mitochondrial proteins, including MDH, were identified. The study suggested a potential phosphorylation site at serine residue 27 (S27) on the MDH protein from S. cerevisiae. This phosphorylation event was found to be regulated in response to oxidative stress conditions.

  Finding the Kinase: Predicting the specific kinase responsible for phosphorylating a particular site on a protein can be challenging but can be aided by several computational and experimental approaches. Here are some commonly used methods:

  Sequence motif analysis: Phosphorylation sites often exhibit sequence motifs that are recognized by specific kinases. Tools like motif analysis algorithms (e.g., ScanSite, GPS, NetPhos) can scan the protein sequence surrounding the phosphorylation site and identify potential kinase recognition motifs. These motifs provide clues about which kinases are likely to phosphorylate the site.

  Phosphosite databases: Databases such as PhosphoSitePlus, Phospho.ELM, or PhosphoNET store information about known phosphorylation sites, including the kinase responsible for their phosphorylation. Researchers can search these databases to identify previously reported kinases for the specific phosphorylation site of interest.

  Kinase prediction algorithms: Several computational tools have been developed to predict potential kinases based on the characteristics of the phosphorylation site and surrounding protein sequence. These algorithms utilize machine learning techniques or sequence-based features to make predictions. Examples include NetworKIN, GPS, or PPSP.

  Experimental kinase assays: In vitro kinase assays can be performed using purified kinases and the target protein or peptide fragment to determine if a specific kinase can phosphorylate the site in question. Radioactive or fluorescent labeling techniques can be employed to detect phosphorylation. Kinase inhibitors or siRNA knockdown experiments can be used to confirm the involvement of specific kinases in cellular contexts.

  Mass spectrometry-based phosphoproteomics: Large-scale phosphoproteomics studies can provide insights into the phosphorylation events occurring within a cellular context. By comparing phosphorylation profiles between different experimental conditions or kinase knockout models, researchers can identify candidate kinases responsible for specific phosphorylation sites.

  It is important to note that kinase prediction methods and experimental assays provide predictions and evidence based on available knowledge, but they do not guarantee definitive identification of the responsible kinase. Integration of multiple approaches, including bioinformatics predictions and experimental validations, is often necessary to gain a comprehensive understanding of kinase-substrate relationships and phosphorylation events.

  It's important to note that the presence of a potential phosphorylation site does not necessarily indicate that phosphorylation occurs under physiological conditions or that it has functional consequences. Further studies are needed to validate and characterize the phosphorylation of MDH at specific sites, including investigating the kinases responsible for the phosphorylation and the functional implications of these modifications.

Information & Resources

PPT Introducing Phosphorylation and MDH CUREs

There are several databases that provide information on phosphorylated proteins. These databases curate and integrate data from various sources, including experimental studies and high-throughput phosphoproteomic analyses. These databases are regularly updated as new phosphorylation events are discovered. It is recommended to explore multiple databases and consider cross-referencing information from different sources to obtain a comprehensive view of phosphorylated proteins. Here are some commonly used databases to find phosphorylated proteins:

• PhosphoSitePlus: PhosphoSitePlus (https://www.phosphosite.org) is a comprehensive database that contains information on experimentally verified phosphorylation sites from various species. It provides detailed annotations, including the kinase responsible for the phosphorylation, protein domains, functional consequences, and associated diseases.

• Phospho.ELM: Phospho.ELM (http://phospho.elm.eu.org) is a database that focuses on experimentally determined phosphorylation sites. It provides curated information on phosphorylation events, including protein kinases, protein-protein interactions, and structural context.

• UniProt: UniProt (https://www.uniprot.org) is a widely used protein database that provides information on protein sequences, functions, and post-translational modifications, including phosphorylation. UniProt contains both manually curated and computationally predicted phosphorylation sites.

• dbPTM: The dbPTM database (http://dbptm.mbc.nctu.edu.tw) integrates information on various post-translational modifications, including phosphorylation. It includes experimentally validated as well as predicted phosphorylation sites from multiple sources.

• PhosphoPep: PhosphoPep (http://www.phosphopep.org) is a database specifically focused on human phosphorylation events. It provides information on phosphorylation sites identified in various tissues, diseases, and experimental conditions.

• Human Protein Reference Database (HPRD): HPRD (http://www.hprd.org) is a resource that contains information on protein-protein interactions, post-translational modifications, and disease associations. It includes phosphorylation data from multiple sources.

Relevant Publications

• Phosphorylation of the Transit Sequence of Chloroplast Precursor Proteins (Waegemann JBC 1996)

• Biochemical Analysis of the NAD+-Dependent Malate Dehydrogenase, a Substrate of Several Serine/Threonine Protein Kinases of Mycobacterium tuberculosis (Wang PLoS 2015)

• Three glycolytic enzymes are phosphorylated at tyrosine in cells transformed by Rous sarcoma virus (Cooper Nature 1983)

List of Skills/ Techniques Needed:

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A list of adopters: Amy Parente, Celeste Peterson, Amy Springer, Joseph Provost… (email josephprovost@sandiego.edu to add your name!)