Kinetic characterisation of the enzymatic activity of the eEF‐2‐specific Ca²⁺‐ and calmodulin‐dependent proteinkinase III purified from rabbit reticulocytes

Abstract

The Ca2+-and calmodulin-dependent protein kinase 111, which specifically phosphorylates the eukaryotic elongation factor 2 (eEF-2), has been purified to apparent homogeneity from the post-ribosomal fraction of rabbit reticulocytes by an efficient four-step method. The method results in a more than 4000-fold purification of the enzyme. SDS-gel electrophoresis showed that the purified kinase contained only one polypeptide with the apparent molecular mass of 90 kDa. The kinase activity was associated with the 90-kDa protein as shown by analyzing the phosphorylating activity of SDS gel electrophoretically purified protein electroblotted to nitrocellulose membranes. The purified kinase was dependent on Ca2+, Mg2+ and calmodulin for activity. Kinetic analysis of the phosphorylation reaction indicates that the turnover number of the kinase was approximately 1 s-'. The K, for the two substrates ATP and eEF-2 was calculated to be approximately 100 pM and 10 pM, respectively. The activity of the kinase was competitively inhibited by CAMP. The inhibition constant Ki (0.5 mM) was found to be in the same order of magnitude as that calculated for the competitive product inhibition caused by ADP. GTP was ten-times less efficient as competitor, indicating that the kinase had a preference for adenosine nucleotides. Phosphorylation of eEF-2 did not interfere with the diphtheria-toxin-catalysed ADP-ribosylation of the factor nor did ADP-ribosylation inhibit phosphorylation.Reversible protein phosphorylation is a common and important mechanism for regulation of biochemical processes such as cell motility, release of neurotransmitters, glycogen metabolism, gene activity and protein synthesis [l]. The activity of the kinases responsible for these protein phosphorylations are regulated by different mechanisms. The best characterised protein kinases are regulated by cyclic nucleotides such as cAMP and cGMP, or by Ca2+ 12, 31. In the latter case enzyme activity is also dependent on a phospholipid or the calcium-binding protein calmodulin Several Ca2+/CaM-dependent protein kinases are known [2]. This group of enzymes includes phosphorylase kinase, myosin light chain kinase, Ca2 +/CaM-dependent protein kinases I, I1 and 111. Ca2 +/CaM protein kinase I11 is a recently discovered kinase that specifically phosphorylates eEF-2 [4 -lo], the eukaryotic elongation factor catalyzing the translocation of peptidyl-tRNA from the ribosomal A-site to the Psite in the protein synthesis elongation cycle [ll].The extent of eEF-2 phosphorylation in vzvo is increased after treatment of cells with drugs that raise the intracellular level of Ca2+, such as thrombin, histamine and veratridine [lo, 121. Phosphorylation of eEF-2 both in vivo and in vitro is correlated with an inhibition of the protein synthesis as a result of a reduced affinity of the modified factor for ribosomes in the pre-translocation phase of the elongation cycle [6, 8, 13, ( c a w ~3 1 .Correspondence to 0. Nygird, Department of Cell Biology, Biology E5, Stockholm University, S-106 91 ...

Interaction of the calcium and calmodulin regulated eEF‐2 kinase with heat shock protein 90

“…These results are unexpected as no Ca" and CaM-independent eEF-2 kinase activity has been observed in reticulocyte lysates [7]. Although phosphorylation of eEF-2 using partially purified kinase preparations [9,22] or the hsp 90 free kinase is strictly dependent on Ca2' and CaM (not illustrated) it has recently been reported that purified preparations of CaM PK III can be converted into a Ca" and CaMindpendent form presumably by autophosphorylation [ 10,111. This may indicate that a similar activation procedure occurs during isolation of the kinase from reticulocyte lysates using the 8D3 anti-hsp 90 mAb immunoadsorbent.…”

“…Rabbit reticulocyte lysates and eEF-2 were prepared as previously described [20,21]. The eEF-2 kinase (CaM PK III) was purified from rabbit reticulocyte lysates essentially as previously described [22].…”

Kinetics of calcium and calmodulin‐dependent protein kinase III from embryonic chicken leg muscle cells

“…The K m and V max for the phosphorylation reaction were calculated to 145±10 μM and 4.5±0.2 pmol/min, respectively. The K m is comparable to that observed for the partially purified kinase from RRL [17]. Based on these results, the ATP concentration used in the following experiments was selected to allow maximum CaM PKIII activity.…”

Delayed polarization of mononuclear phagocyte transcriptional program by type I interferon isoforms

“…Signature IFNs specifically affected a group of genes involved in eukaryotic translation and listed in category 13) biosynthesis/regulation of translation: eukaryotic translation initiation factor 5A (EIF5A) [63], eukaryotic translation elongation factor 1 delta (EEF1D) [64], and eukaryotic translation elongation factor 2 (EEF2) [65]. This supports the notion that the powerful stimulus of activation induced by the signature IFNs on immune cells is a global type of regulation directly affecting target molecules at the levels of both transcription and translation.…”