Death-associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis

Abstract

Death-associated protein kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that is a drug discovery target for neurodegenerative disorders. Despite the potential profound physiological role of DAPK in neuronal function and pathophysiology, the endogenous substrate(s) of this kinase and the mechanisms via which DAPK elicits its biological action remain largely unknown. We report here that the mammalian 40S ribosomal protein S6 is a DAPK substrate. Results from immunoprecipitation experiments are consistent with endogenous DAPK being associated with endogenous S6 in rat brain. When S6 is a component of the 40S ribosomal subunit complex, DAPK selectively phosphorylates it at serine 235, one of the five sites in S6 that are phosphorylated by the S6 kinase family of proteins. The amino acid sequence flanking serine 235 matches the established pattern for DAPK peptide and protein substrates. Kinetic analyses using purified 40S subunits revealed a K(m) value of 9 microM, consistent with S6 being a potential physiological substrate of DAPK. This enzyme-substrate relationship has functional significance. DAPK suppresses translation in rabbit reticulocyte lysate, and treatment of neuroblastoma cells with a stimulator of DAPK reduces protein synthesis. In both cases, suppression of translation correlates with increased phosphorylation of S6 at serine 235. These results demonstrate that DAPK is a S6 kinase and provide evidence for a novel role of DAPK in the regulation of translation.

Figure 1. Mammalian ribosomal protein S6 contains a putative DAPK phosphorylation site and interacts with DAPK in rat brain

(A) The sequence of the human ribosomal protein S6 contains a substrate phosphorylation pattern preferred by DAPK (underscore) in which Ser235 (*) is the putative phosphorylation site. Basic residues, known to be favored in DAPK substrates upstream of the phosphorylation site, are indicated (double underscore). Other sites in ribosomal protein S6 known to be phosphorylated by S6 kinases, Ser236 (‡), Ser240 (π), and Ser244 (+), are also indicated. (B) Endogenous DAPK was immunoprecipitated from rat brain. The immunoprecipitate was analyzed by Western blotting for DAPK and ribosomal protein S6. DAPK immunoprecipitate (lane 1); control immunoprecipitate in the absence of DAPK antibody (lane 2). Data are representative of 2 independent experiments.

Figure 2. Mammalian ribosomal protein S6 is a DAPK substrate

(A) Rat liver 40S ribosomal subunits were phosphorylated in vitro by DAPK and analyzed by Western blotting with antibodies against phosphorylated Ser235, phosphorylated Ser235/Ser236, or phosphorylated Ser240/Ser244 epitopes. Increased phosphorylation of the Ser235 and Ser235/Ser236 epitopes, but not of the Ser240/Ser244 epitope, was observed in the presence of DAPK. Equal levels of ribosomal protein S6 were confirmed by blotting for total S6 protein. Results are representative of 2 independent experiments. (B) Constitutively active DAPK was used to phosphorylate 40S ribosomal subunits. Concentrations of 40S ranging from 1.25 μM to 10 μM were used. The K_m was determined by measuring ³²P-incorporation as described in Materials and Methods. Error bars represent mean values ± SEM, and are not shown if the error is smaller than the symbol. r²=0.95 for the linear regression analysis. Phosphorylation of ribosomal protein S6 was verified by phosphoimaging (inset).

Figure 3. Phosphorylation of ribosomal protein S6 at Ser235 by DAPK does not affect downstream phosphorylation at Ser240/Ser244 by p70 S6 Kinase

Small ribosomal subunits from rat liver were treated with alkaline phosphatase and incubated with either DAPK, or p70 S6K, or DAPK and p70 S6K. The total amount of ribosomal protein S6, and the amount of its phosphorylated forms at Ser235 and Ser240/Ser244 was identified by Western blotting. DAPK-treated 40S subunits displayed phosphorylation at Ser235; p70 S6K-treated 40S subunits showed phosphorylation at Ser240/Ser244; and 40S subunits that had been phosphorylated by DAPK and p70 S6K showed elevated levels of phosphorylation at Ser235 as well as at Ser240/Ser244. Results are representative of 2 independent experiments.

Figure 4. DAPK inhibits translational activity

(A) The effect of DAPK on translation in vitro was determined using rabbit reticulocyte lysate. DAPK was incubated with rabbit reticulocyte lysate prior to the addition of human liver mRNA and the effect of the kinase activity on translation was quantified by measuring [³⁵S]-Methionine incorporation into newly synthesized peptides. Data is expressed as a percentage of the maximum translation activity and is the mean ± SEM of 5 replicates from 2 independent experiments. P < 0.05 (*). (B) DAPK phosphorylates ribosomal protein S6 at Ser235 within the rabbit reticulocyte lysate. Samples of the reticulocyte lysate incubated in the presence or absence of DAPK were subjected to Western analysis. Equal amounts of reticulocyte lysate were present in each sample. Data shown are representative of two independent experiments. (C) The effect of ceramide, a stimulator of DAPK, on protein synthesis and ribosomal protein S6 phosphorylation were determined in SH-SY5Y neuroblastoma cell cultures. Incorporation of [³⁵S]-Methionine after 1 h treatment of cells with ceramide was quantified. Data is the mean ± SEM and is representative of 2 independent experiments. P = 0.001 (*). (D) Phosphorylation of ribosomal protein S6 at Ser235 in response to 1 h treatment with ceramide was determined by Western blotting. Equal protein in each lane was verified by blotting for ribosomal protein S6.

Figure 5. Phosphorylation of S6 by DAPK is directly involved in the inhibition of translation

(A) Schematic representation of the fractionation protocol followed to dissect the inhibitory effect of DAPK on protein synthesis (for details, see Materials and Methods). (B) Phosphorylation of ribosomal protein S6 at Ser235 following treatment of salt-washed ribosomes with DAPK was determined by Western blotting. Equal protein in each lane was verified by blotting for ribosomal protein S6. (C) Translational activities of rabbit reticulocyte reconstituted systems. Supernatant at 100,000xg (S100) and ribosomal salt wash (RSW) fractions were supplemented before the translation reaction with ribosomes treated with either no kinase (control) or DAPK. Data shown are representative of three independent experiments.