Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation

Abstract

Eukaryotic elongation factor 1 (eEF-1) contains the guanine nucleotide exchange factor eEF-1B that loads the G protein eEF-1A with GTP after each cycle of elongation during protein synthesis. Two features of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the significance of target sites for the cell cycle protein kinase CDK1/cyclin B. The roles of these two features were addressed by elongation measurements in vitro using cell-free extracts. A poly(GUA) template RNA was generated to support both poly(valine) and poly(serine) synthesis and poly(phenylalanine) synthesis was driven by a poly(uridylic acid) template. Elongation rates were in the order phenylalanine > valine > serine. Addition of CDK1/cyclin B decreased the elongation rate for valine whereas the rate for serine and phenylalanine elongation was increased. This effect was correlated with phosphorylation of the eEF-1delta and eEF-1gamma subunits of eEF-1B. Our results demonstrate specific regulation of elongation by CDK1/cyclin B phosphorylation.

Figure 1

Elongation of amino acids from template RNAs. Poly(phenylalanine) was determined using poly(uridylic acid) as template and poly(valine) and poly(serine) synthesis were determined using poly(GUA) as template. Reactions were initiated by radioactive amino acid addition in a total volume of 110 µl at 30°C. Aliquots of 10 µl were taken at the indicated times for the determination of elongation in c.p.m.

Figure 2

Effect of CDK1/cyclin B on elongation. Elongation rates were determined as indicated in Materials and Methods for a 1 h incubation at 30°C. Columns represent elongation in the presence of the indicated component, expressed as percent of the corresponding control. Bars represent the standard errors of four determinations in the same experiment.

Figure 3

CDK1/cyclin B activity under the translation assay conditions. Autoradiography of ³²P-labelled proteins after incubation as indicated in Materials in Methods and resolution by SDS–PAGE. Each lane corresponds to the indicated incubation conditions. Left, molecular weight markers are indicated in kDa.

Figure 4

Phosphorylation of eEF-1B under the translation assay conditions of the lysates. Western blot and corresponding autoradiography of the eEF-1B complex immunoprecipitated from the lysate after incubation with [³²P]ATP, as indicated in Materials and Methods. (Left) Western blot performed with anti-eEF-1β antibody from incubations performed without (–) or with (+) CDK1/cyclin B. (Right) Corresponding autoradiogram. Left, molecular weight markers are indicated in kDa.