All living organisms must constantly monitor their internal and external environment to sense signals for growth, development and reproduction.
Transcription of new mRNAs, alternative RNA splicing and translation of new proteins create a continuously fine-tuned regulatory network to adapt to environmental alterations. A major mechanism to directly respond to changing circumstances without expanding the size of the genome or varying the cellular gene expression is represented by posttranslational protein modifications (PTMs). PTMs increase the functional diversity of protein variants by adding chemical groups or other proteins to one or more of its amino acid residues. Often, multiple residues are modified on the same protein. This may happen through the same type of modification on various sites as well as through different types of modifications on multiple, or overlapping sites (Prabakaran et al, 2012). Multiple PTMs lead to an exponential increase of potential molecular states. These modifications alter protein surfaces, lead to differences in the interaction with other proteins and can affect stability, activity or localization of targeted substrates (Mann & Jensen, 2003; Duan
& Walther, 2015). PTMs control a wide range of cellular pathways including development, cell-cycle control, DNA repair, cell growth and signaling (Seo & Lee, 2004; Deribe et al, 2010; Chung & Dellaire, 2015). They can vary in a temporal and spatial manner depending on the respective environmental condition. Defects in posttranslational protein modifications have been linked to a variety of diseases and developmental disorders demonstrating the importance of PTMs in maintaining normal cell viability (Karve & Cheema, 2011).
More than 200 kinds of PTMs are known to modify eukaryotic proteins (Walsh & Jefferis, 2006). Several were discovered years ago and their broader significance has emerged only slowly. Some PTMs such as glycosylation, lipidation and disulfide bridge formation are stable and have essential roles in maturation and proper folding of newly synthesized proteins. Others, including phosphorylation, sumoylation, ubiquitination and neddylation are reversible and are important for
dynamically regulated network. Analyses of these modifications and participating enzymes are challenging due to their transient occurrence, but they provide indispensable insights into biological functions of proteins and increase the possibilities to develop therapeutic proteins (Walsh & Jefferis, 2006; Pratt et al, 2015).
Figure 1: Posttranslational modifications of proteins.
After protein synthesis, posttranslational modifications (PTMs) increase the diversity of the cellular proteome without altering the transcriptome. The attachment of functional groups or proteins can change the function of targeted substrates. PTMs include phosphorylation, glycosylation, ubiquitination, neddylation, as well as sumoylation and can affect the stability, activity, interaction or localization of the modified target protein.
1.1.1 Phosphorylation and dephosphorylation
Phosphorylation was one of the first posttranslational modifications described (Fischer & Krebs, 1955). Phosphorylation of cellular proteins is a dynamic process that depends on the activity of protein kinases and protein phosphatases (Hunter, 1995). A kinase catalyzes the transfer of -phosphate from ATP to its protein substrate, whereas a phosphatase removes the phosphate by hydrolysis (Figure 2).
Although kinase encoding genes constitute 2% of eukaryotic genomes, they phosphorylate more than 30% of all cellular proteins (Ubersax & Ferrell, 2007).
Since kinases and phosphatases have severe impacts on their substrates, the recognition and interaction with the target protein has to be precisely regulated and the protein modification must be strictly controlled in a specific temporal order
INTRODUCTION
(Rogers et al, 2015). Kinases and phosphatases can either identify their substrates by a specific consensus sequence of the phosphorylation site in the substrate or by interaction motifs spatially separated from the modified residue. Additional levels of substrate specificity are provided by the amino acid composition of the respective catalytic core and the co-localization of kinases/phosphatases with their particular substrates in the same cellular compartment (Ubersax & Ferrell, 2007; Cheng et al, 2011). The addition of a phosphate group occurs in eukaryotes primarily on the amino acid residues of serine, threonine and tyrosine, which adds two negative charges to the substrate. This can lead to conformational changes affecting the biological function of the protein (Nishi et al, 2011; Duan & Walther, 2015).
Figure 2: Mechanism of phosphorylation and dephosphorylation of proteins.
Proteins can be phosphorylated on serine/threonine side chains (dashed blue circle) by serine/threonine specific kinases and dephosphorylated by serine/threonine phosphatases. The reaction involves ATP as the phosphate donor in the phosphorylation reaction and water to hydrolyze the phosphate group (dashed red circle) in the dephosphorylation reaction. This results in a changed structure, activity or stability of the substrate (illustrated in a simplified form as a change of the grey periphery).
Due to different amino acids that can be modified by a phosphate group, two major families of kinases and phosphatases exist. One group is specific for tyrosine side chains and another one targets serine/threonine residues. Phosphorylation and
2005; Barr et al, 2011). Around 98% of reversibly protein-bound phosphate in eukaryotic cells affects serine/threonine residues (Olsen et al, 2006). Serine/threonine phosphatases can be divided into the family of phosphatases metallo-dependent (PPM) and the phospho-protein phosphatases (PPPs). Both types of enzymes contain a conserved domain with an active site consisting of two metal ions, such as manganese (Mn2+) and iron (Fe2+), which are surrounded by a set of conserved amino acid residues (Shi, 2009). These bound metal ions are crucial to coordinate the phosphorus of the substrate and to stabilize its negative charge, thus facilitating nucleophilic attack on the phosphate group by a water molecule and hydrolysis of the phosphate ester bond (Goldberg et al, 1995; Barr et al, 2011).