As eEF2K regulates a key player in protein translation it seems reasonable to begin with a brief overview of the steps involved to set the stage for the next sections in the literature review.
Protein translation is by definition the conversion of the degenerate triplet code found on mRNA into its corresponding amino acid sequence. mRNA sequences are decoded in the 5' to 3' direction while the polypeptide is synthesised from its N-terminus to C-terminus. Two highly conserved ribonucleoprotein subunits: a small 40S and large 60S subunit (in eukaryotes) that come together to create the functional translation machinery, the ribosome. When assembled the ribosome has three binding sites for tRNA. The A site 'accepts' incoming aminoacyl-tRNAs to be added to the growing polypepide chain attached to the tRNA in the P site. The E site is the location of the deacylated tRNA before its release form the ribosome. Fidelity in translation is vital and is brought about by the ribosome and its cofactors in three controlled steps:
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Initiation: This requires the assembly of mRNA, tRNA corresponding to the start codon and the ribosome. The 'Shine-Dalgarno' sequence just upstream of the start codon is recognised by the 40S subunit which positions the start codon correctly in the P site. fMet-tRNA contains the CAU anticodon necessary to bind the start codon. Unlike all other aminoacyl-tRNAs it enters through the P site not the A due to its formyl modification. Initiation is aided by initiation factors 1,2 & 3. IF1 and IF3 are involved in stabilising the 40S initiation complexes while IF2 promotes the association of fMet-tRNA with the 40S subunit.
Elongation: With fMet-tRNA in the P site, the incoming aminoacyl-tRNA is translocated to the A site with help from GTPase EF1a. Once the tRNA is in place ribosome mediated GTP hydrolysis occurs followed by release of GDP-bound EF1a. Nucleophilic attack on the fMet-tRNA occurs by aminoacyl-tRNA in the A site. The condensation reaction produces a peptide bond between the two amino acids which are joined to the tRNA in the A site.
Large structural rearrangements within the ribosome take place which move deacylated tRNA in P site to E site and peptidyl-tRNA in A site to P site. This process is aided by GTPase EF2 and for obvious reasons, explained in further detail in later sections. The A site is now empty and ready to accept another aminoacy-tRNA to start the process over.
Termination: Release factors are responsible for the recognition of a stop codon within the A site. They trigger cleavage of the polypeptide from P site tRNA and assist in polypeptide release from ribosome.
Eukaryotic Elongation factor 2
While there are a number of factors that control elongation at the translational level only eEF2 will be discussed here. eEF2 is homologous both in structure and function to its bacterial and archeal counterparts (EF2G and aEF2 respectively). It also displays extremely high sequence conservation (over 99%) within the mammalian kingdom.(1)
eEF2 is a cytoplasm-bound monomeric protein comprised of 857 amino acid residues. Within the 95.2kDa protein are six structural domains (I-VI) that are change in relative orientation upon ribosome binding and GTP hydrolysis.(2)
Its role in the protein synthesis pathway is to aid the translocation of tRNAs in the A and P site of the 80S ribosome to the P and E sites respectively, a distance of over 20 Å (3). Cryo EM studies show conformational changes of eEF2 upon ribosome binding (4). Domains III-V shift with a hinge-like manner relative to domains I & II, which propogate to the ribosome to induce a ratchet-like motion of the small subunit with respect to the 60S subunit(4). These conformational changes account for the movement of the anticodon stem loop of the tRNA in the A site and the decoding centre of the large subunit towards site P in the direction of site P. Domain IV of eEF2 is then able to bind to site A which changes the contact points between the ribosomal subunits (4).
GTP hydrolysis of eEF2 causes conformational changes including a 6Å shift in domin IV that ultimately results in the breaking of string contact between the codon-anticodon stem loop of the A site tRNA and the decoding centre. Head movement of the large subunit is no longer impeded and its rotation moves the anticodon stem loop of the tRNAs the remaining distance required for full translocation which subsequently occurs. eEF2-GTP is then free to leave the ribosome which allows the head and small subunit to move back to their original positions.
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Regulation of eEF2: the how
eEF2 is negatively regulated by phosphorylation which results in its inactivation. Activated eEF2K phosphorylates eEF2 on specific threonine, tyrosine and serine residues found towards the N-terminus of eEF2. Phosphorylation of Thr58 can only take place after phosphorylation on Thr56 and these two sites are the most important. (shitty needs work). Phosphorylation of Thr56 results in loss of ribosome binding rendering eEF2 inactive. This also hints that the ribosome binding site is located within the N-terminus of eEF2. The C-terminus is also believed to interact with the ribosome(Nygard Nilsson 1990[25 in browne 2002]).
A feature unique to eEF2 is a diphthamide (post-translationally modified histidine) residue which acts as an allosteric site for cellular enzymes inhibiting eEF2 through transfer of an adenosine diphosphate ribosyl moiety from an NAD+ molecule.(Taylor 2007)
The covalent modifications resulting eEF2 inactivation are reversed by cellular phosphatases namely protein phosphotase 2A (PP2A).
Regulation of eEF2: the why
Protein synthesis requires a vast amount of the available cellular energy and a large proportion of this is consumed during elongation. This is due to 4 high energy bonds being broken with each amino acid added to the incipient chain; two from the AMP generated through the formation amino acyl-tRNA (AMP requires an ATP molecule to convert it to ADP, therefore 1AMP = 2ADP) and further 2 GDP molecules are generated from GTP on the ribosome through action of elongation factors. So when cellular energy needs to be conserved, in times of increased energy demand or decreased energy supply, it follows that the elongation step be tightly regulated. Inhibition of elongation allows the polysome to be kept static, implying protein synthesis can be rapidly resumed and mRNA is kept stable by polysomes. Conversely if just initiation were controlled all the ribosomes on the mRNA would need to reach termination before protein synthesis could be stalled, furthering the cell's energy debt. Also were it just the initiation step that were controlled, a bottleneck would form with many ribosomes competing for attachment to mRNA if the rate elongation could not be increased.
Introduction to Kinase superfamily
When eEF2K was discovered it showed no homology to the two main kinase superfamilies; S/T/Y and H kinase.. It was first classified under CaM-dependant kinse family of proteins due to its dependence on Ca2+/CaM for activity. Ryazanov et al., however showed it has no sequence homology to other CaM-dependent kinases save for a glycine rich motif GXGXXG within the ATP binding site found in all protein kinases. Its catalytic site recognises residues within Î±-helical secondary structures as opposed to the conventional Î²-turn or loops seen in all the other kinases, identifying eEF2K's place within the atypical kinase superfamily as part of a novel family called the alpha kinases.
EEF2K: structure and function
The structure of eEF2K remains elusive but site-directed mutational analysis work done by Pavur 2000, Diggle 1999, has mapped out it's functional domains: an N-terminal catalytic domain (100-335) and a C -terminal eEF2 recognition site (521-725) separated by a linker region (336-520). The CaM binding domain was shown to immediately preceed the catalytic site at the N -terminus (77-99). Removal of even the last 19 amino acids rendered eEF2K unable to bind eEF2 however autophosphorylation could still occur, implying that the extreme C-terminus contains a key eEF2 interaction. A representation of eEF2K's domains is shown in Figure 1.X
Regulation of eEF2K
Negative regulation by PKA and AMPK
PKA (cAMP-dependent Protein Kinase A) phosphorylates eEF2K on serine residue 500 (in eukaryotes), which gives eEF2K the ability to remain constitutively active in the absence of both a Ca2+ signal and CaM (Redpath 1993, Diggle 2001).
Interestingly in heart cells PKA phosphorylation does not activate eEF2K independently of CaM but instead just raised it maximal activity.(47 Browne 2002). As intracellular Ca2+ levels rise and fall from beat to beat in cardiac myocytes it seems more pertinent to increase eEF2K's maximal activity from another pathway rather than rendering it independent to Ca2+.
Another negative regulator of eEF2K is AMPK (AMP activated Protein Kinase). Horman et al. 2002 shows that in times of ATP depletion (by ATP synthesis blocker oligomycin), phosphorylation of eEF2K is increased in liver HEK293 cells. It also showed that the same results aren't observed in cells deficient in functional AMPK.
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As PKA is usually activated during times of increased energy consumption and AMPK is an energy status sensor within cells, their overall regulatory effect on eEF2K within the context of protein translation is clear; their activation in times of energy deficit results in increased phosphorylation levels of eEF2 and the inhibition of elongation of peptide translation, a strategy employed to staunch the high energy depletion of protein synthesis to conserve it for more immediate metabolic needs.
Chinese hamster ovary (CHO) cells overexpressing the insulin receptor have been shown to cause rapid dephosphorylation of eEF2K on exposure to insulin(redpath, proud 1996 [B2002]). These findings have been replicated in adipocytes and ventricular myocytes[50,53 B2002). This implicated a role for eEF2 within the insulin pathway and more work showed that mTOR dependent S6K1 phosphorylates eEF2K at serine 366 rendering it inactive at basal Ca2+ concentration levels(54).
S6K1 however is not the only kinase that phosphorylates eEF2K at S336. Evidence has shown that angiotensin II, phenylephrine and endothelin all decrease eEF2 phosphorylation levels. These all operate through the MAPK pathway and incriminate p90RSK as the final effector of eEFK.(55,56)
Serine 366 is not the only phosphorylation site that deactivates eEF2K, phosphorylation of S359 was shown to occur in response to Insulin-like growth factor 1 (IGF1) which is blaocked by rampamycin implicating mTOR in the signalling cascade. However only stress activated protein kinase 4 (SAPK4) has been demonstrated to phosphorylate S359, so there must be another kinase to be indentified(63). (might just go in pic(Another phosphorylation site within this pathway has also been identified. Phosphorylation at S377 was shown to increase in response to SAPK4 activation 59. This phosphorylation site does not seem to affect eEF2K activity.))