Search

TRPM6 and TRPM7 encode channel-kinases. While these channels share electrophysiological properties and cellular functions, TRPM6 and TRPM7 are non-redundant genes raising the possibility that the kinases have distinct substrates. Here, we demonstrate that TRPM6 and TRPM7 phosphorylate the assembly domain of myosin IIA, IIB and IIC on identical residues. Whereas phosphorylation of myosin IIA is restricted to the coiled-coil domain, TRPM6 and TRPM7 also phosphorylate the non-helical tails of myosin IIB and IIC. TRPM7 does not phosphorylate eukaryotic elongation factor-2 (eEF-2) and myosin II is a poor substrate for eEF-2 kinase. In conclusion, TRPM6 and TRPM7 share exogenous substrates among themselves but not with functionally distant α-kinases.Structured summaryMINT-6700314:GNA1 (uniprotkb:Q96EK6) and GNA1 (uniprotkb:Q96EK6) bind (MI:0407) by X-ray crystallography (MI:0114)

Previously we have found that elongation factor 2 (EF-2) from mammalian cells can be phosphorylated by a special Ca2+/calmodulin-dependent protein kinase (EF-2 kinase). Phosphorylation results in complete inactivation of EF-2 in the poly(U)-directed cell-free translation system. However, the partial function of EF-2 affected by phosphorylation remained unknown. Here we show that phosphorylated EF-2, unlike non-phosphorylated EF-2, is unable to switch ribosomes carrying poly(U) and Phe-tRNA in the A site to a puromycin-reactive state. Thus, phosphorylation of EF-2 seems to block its ability to promote a shift of the aminoacyl(peptidyl)-tRNA from the A site to the P site i.e. translocation itself.

The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3–5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.

There are many reports which demonstrate that the rate of protein biosynthesis at the elongation stage is actively regulated in eukaryotic cells. Possible physiological roles for this type of regulation are: the coordination of translation of mRNA with different initiation rate constants; regulation of transition between different physiological states of a cell, such as transition between stages or the cell cycle; and in general, any situation where the maintenance of a particular physiological state is dependent on continuous protein synthesis. A number of covalent modifications of elongation factors offer potential mechanisms for such regulation. Among the various modifications of elongation factors, phosphorylation of eEF-2 by the specific Ca2+/calmodulin-dependent eEF-2 kinase is the best studied and perhaps the most important mechanism for regulation of elongation rate. Since this phosphorylation is strictly Ca2+-dependent, and makes eEF-2 inactive in translation, this mechanism could explain how changes in the intracellular free Ca2+ concentration may regulate elongation rate. We also discuss some recent findings concerning elongation factors, such as the discovery of developmental stage-specific elongation factors and the regulated binding of eEF-1α to cytoskeletal elements. Together, these observations underline the importance of the elongation stage of translation in the regulation of the cellular processes essential for normal cell life.

Incubation of a ribosome-free extract of rabbit reticulocytes or rat liver with [gamma-32P]ATP and Ca2+ results in incorporation of 32P predominantly into a single polypeptide of Mr approximately 100,000. This polypeptide is identified as elongation factor 2 (EF-2). Phosphorylation of EF-2 is strictly Ca2+-dependent and can be inhibited by the calmodulin antagonist trifluoperazine. It is suggested that the Ca2+/calmodulin-dependent phosphorylation of EF-2 is involved in regulation of protein biosynthesis.

eEF-2 kinase is a ubiquitous Ca2+/calmodulin-dependent protein kinase that is specific for protein synthesis elongation factor-2 (eEF-2). This study describes an improved procedure for the purification of eEF-2 kinase from rabbit reticulocyte lysate. The eEF-2 kinase preparation was used to raise polyclonal antibodies, which immunoprecipitated eEF-2 kinase protein and activity from rabbit reticulocyte lysate. The antibodies recognized a single 103 kDa band in extracts from several cell lines including NIH 3T3, PC12, C6 glioma, HeLa, and MCF-7 breast carcinoma. However, there was no immunoreactivity in extracts of rabbit or bovine liver or rabbit kidney despite the presence of abundant eEF-2 kinase activity in these tissues. Exposure of PC12 cells to nerve growth factor (NGF) resulted in rapid down-regulation of eEF-2 kinase activity and a decrease in immunoreactivity. After 24 h of incubation with NGF, the activity of the kinase recovered to 80% of initial values. In contrast, the immunoreactivity of eEF-2 kinase continued to decrease. These data suggest that tissue-specific isoforms of eEF-2 kinase may exist and that these isoforms may be regulated by growth factors.

A model is proposed uniting two groups of facts: The adsorption of enzymes on subcellular structures and the direct (‘from hand to hand’) transfer of metabolites between enzymes. The basic idea is that the binding of metabolites (substrates and/or products) results in desorption of the enzymes from subcellular structures during each catalytic act. This makes the enzymes mobile and capable of directly (from hand to hand) transferring metabolites to other enzymes adsorbed on subcellular structures. The model leads to a mechanism by means of which soluble enzymes can be compartmentalized in defined regions of the cytoplasm.

Several aminoacyl-tRNA synthetases of higher eukaryotes have always been found as multienzyme complexes. There are indications that these complexes can be associated with some tRNA-modifying enzymes. The function of such complexes is unclear. I have noticed that 6 out of 7 aminoacyl-tRNA synthetases most commonly occurring in complexes correspond to a group of tRNAs which must always contain a modified U in the first position of their anticodons. A hypothesis is proposed according to which association of 6 aminoacyl-tRNA synthetases with U-modifying enzymes can protect a cell from miscoding.

The nerve growth factor-sensitive phosphoprotein from PC12 cells, previously designated Nsp100, has been shown to be elongation factor 2 (EF-2). The criteria used for this identification include: (i) similarity of N-terminal sequence; (ii) phosphorylation by the same kinase; (iii) ADP-ribosylation mediated by diphtheria toxin; (iv) comparable function in cell-free protein synthesis. According to these criteria, Nsp 100 and EF-2 are identical and the kinase that phosphorylates Nsp100 in PC12 cells is calcium/calmodulin kinase III.