Presentación del diagnóstico a la Dirección

Phosphorylation of Serine (Ser), Threonine (Thr), Tyrosine (Tyr) and Histidine (His) residues is the best known and most ubiquitous PTM. Because the phosphate group (-O-PO32-) carries a large negative charge at neutral pH, phosphorylation of neutral amino acids induces altered conformations in local protein microenvironments (Johnson and Lewis, 2001). Phosphorylation can therefore modulate protein function by altering protein stability, cellular localization, substrate affinity, complex formation and activity.

The addition of a phosphate group on a substrate protein is catalyzed by protein kinases, while hydrolyses of the phosphate group is achieved by protein phosphatases. Eukaryotes express a large variety of protein kinases (Manning et al., 2002) and phosphatases (Moorhead et al., 2007; Virshup and Shenolikar, 2009), each with a unique substrate specificity and regulation. The expansion of the protein kinase families in higher metazoans accounts for the observed cellular and functional diversity between these organisms. Due to its reversible and transient nature, phosphorylation allows signal transduction pathways to carry out diverse cellular functions. Moreover it also allows essential events such as cell cycle and growth to occur at precise times and locations.

The eukaryotic cell cycle is an example of cellular decision- making based on reversible phosphorylation and dephosphorylation of proteins. One third of the Drosophila kinome was shown to affect cell

cycle progression (Bettencourt-Dias et al., 2004). Cdks regulate the G1, S and G2 phases of the cell cycle ensuring DNA duplication and segregation of chromosomes into daughter cells (fig. 8). Cdks are themselves regulated and cooperate with other protein kinases.

Figure 8: Mitotic phosphoregulation in animal cells. Upper panel shows the different mitotic stages and highlights major events known to be regulated by reversible protein phosphorylation. Middle panel indicates the activity phases of the major mitotic kinases. Lower panel shows known mitotic phosphatases with respect to the depicted mitotic events. Exact timing of activation and inactivation of most mitotic phosphatases is not known as depicted by open bars. (Adapted from Bollen et al., 2009).

Among the different kinases, Cdk1 has an essential role in mitotic entry and progression until all chromosomes are properly aligned along the metaphase plate (Morgan, 2007). Entry into anaphase and subsequent mitotic exit requires inactivation of Cdk1 and other mitotic regulators (Sullivan and Morgan, 2007). This inactivation is primarily mediated by proteosome-dependent degradation of proteins by the APC/C and also by controlled removal

of mitotic phosphorylations by different phosphatases. Mitosis relies therefore in a delicate balance between the activities of kinases and their counteracting phosphatases.

2.1.2. Acetylation

The role of acetylation has been suggested to be analogous to that of phosphorylation (Kouzarides, 2000). Many proteins are posttranslationally acetylated, and at least for eukaryotic proteins, acetylation is the most common covalent modification out of over 200 reported types. Protein acetylation is catalyzed by a variety of different acetyltransferases. These enzymes catalyze the transfer of an acetyl group from acetyl-coenzyme A (Acetyl-CoA) to either the -amino group of amino-terminal residues (N_{-terminal acetylation, revised in} Chpater I, section 3.) or to the-amino group of lysine (Lys) residues at various positions. The-amino group designates the position of the central carbon atom of amino acids, whereas the -amino group of lysine residues designates the position of a carbon atom on the side chain.

The introduction of acetyl groups on Lys side chains potentially converts cationic side chains into neutralized surfaces influencing protein function or its association with other proteins. Acetylation of - Lys residues occurs on histones, high mobility group (HMG) proteins, transcription factors, nuclear receptors (Bannister et al., 2000; Imhof et al., 1997; Roth et al., 2001) and-tubulin (MacRae, 1997).

Most studied acetylated proteins include N_{-acetylation of Lys} side chains on the N-terminal tail of histones (Yang, 2004). Two groups of enzymes responsible for regulating the reversible and dynamic state of histone acetylation are histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation of histones, in

general, leads to transcriptional activation by inducing the unpacking of the nucleosomes from the tight 30nm chromatin fibers. This allows manipulating the tightly packed heterochromatin to more relaxed euchromatin state (fig. 9), which in turn allows other transcriptional regulatory proteins to gain access to promoter elements on DNA. The acetylation state of different promoters is maintained by specific combinations of HATs and HDACs. Not surprisingly, histone acetylation appears to influence other processes including cell cycle progression, chromosome dynamics, DNA replication, recombination and repair, silencing and apoptosis (Kouzarides, 1999).

Figure 9: Histone acetylation and transition of heterochromatin to euchromatin. Representation of the molecular structure of acetylated and deacetylated lysine residues and resultant chromatin state. (adapted from Khan and Khan, 2010).

2.1.3. Methylation

Although there are known examples of protein side chain methylation, histone N-terminal tail methylation draws most research attention. Since methyl groups are relatively small, addition of these groups to Lys and Arginine (Arg) residues does not neutralize their charges, having little effect on histone conformation. Instead, methylation has been associated with the creation of binding sites for

specific regulatory proteins (Bannister and Kouzarides, 2005). Therefore, unlike acetylation, which generally correlates with transcriptional activation, histone lysine methylation can signal either activation or repression, depending on the sites of methylation (Cuthbert et al., 2004). The -amino group of Lys can accept up to three methyl groups and hence can be mono-, di- or trimethylated (Martin and Zhang, 2005). For certain processes, methylation on the same site can lead to different outcomes depending on the number of methyl groups added.

2.1.4. Ubiquitination

Ubiquitination is likely to affect all proteins at some point in their life cycle. Ubiquitin (Ub) a highly conserved 8.5 kDa protein that becomes covalently attached to Lys residues of target proteins in an inducible and reversible manner. This attachment occurs through a 3 step process involving 3 different types of enzymes, E1, E2 and E3 ligases (Pickart and Eddins, 2004). According to the number of Ubs added, substrate proteins can be monoubiquitinated or polyubiquitinated. Monoubiquitination is believed to serve as a regulatory modification of a target protein in much the same way phosphorylation regulates protein activity (Weissman, 2001). However, the most common role associated to ubiquitination is the tagging of proteins for degradation. The vast majority of proteins (80-90%) tagged by Ub polypeptides are then degraded via the 26 S proteasome (Craiu et al., 1997; Rock et al., 1994). This degradation signal usually involves protein polyubiquitination. One example where ubiquitination plays a central role is in cell cycle progression. The characteristic temporal control of proteins destroyed during the cell cycle, such as the cyclin subunits of cdks, is performed by the E1-E2-E3 ubiquitin ligase machinery of the APC/C (Nakayama and Nakayama, 2006).