Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3

Abstract

Mammalian mitochondrial ribosomes synthesize 13 proteins that are essential for oxidative phosphorylation. In addition to their role in protein synthesis, some of the mitochondrial ribosomal proteins have acquired functions in other cellular processes such as apoptosis. Death-associated protein 3 (DAP3), also referred to as mitochondrial ribosomal protein S29 (MRP-S29), is a GTP-binding pro-apoptotic protein located in the small subunit of the ribosome. Previous studies have shown that phosphorylation is one of the most likely regulatory mechanisms for DAP3 function in apoptosis and may be in protein synthesis; however, no phosphorylation sites were identified. In this study, we have investigated the phosphorylation status of ribosomal DAP3 and mapped the phosphorylation sites by tandem mass spectrometry. Mitochondrial ribosomal DAP3 is phosphorylated at Ser215 or Thr216, Ser220, Ser251 or Ser252, and Ser280. In addition, phosphorylation of recombinant DAP3 by Protein kinase A and Protein kinase Cd at residues that are endogenously phosphorylated in ribosomal DAP3 suggests both of these kinases as potential candidates responsible for the in vivo phosphorylation of DAP3 in mammalian mitochondria. Interestingly, the majority of the phosphorylation sites detected in our study are clustered around the highly conserved GTP-binding motifs, speculating on the significance of these residues on protein conformation and activity. Site-directed mutagenesis studies on selected phosphorylation sites were performed to determine the effect of phosphorylation on cell proliferation and PARP cleavage as indication of caspase activation. Overall, our findings suggest DAP3, a mitochondrial ribosomal small subunit protein, is a novel phosphorylated target.

Protein folding creates structure-based, noncontiguous consensus phosphorylation motifs recognized by kinases

“…3B). Death-associated protein 3 (DAP3) (28) is also phosphorylated by PKA and has phosphorylated Ser 185 (P0) and anchoring residues Lys 192 and Lys 189 (Fig. 3C).…”

Chronic ethanol feeding causes depression of mitochondrial elongation factor Tu in the rat liver: implications for the mitochondrial ribosome

“…As with NDUFB8, a depression in DAP3 levels may allow the survival of dysfunctional mitochondria in alcoholic animals, this time by decreasing their ability to undergo apoptosis. The precise role that DAP3 plays within the 28S subunit has yet to be determined, but it is known to bind GTP and have a number of phosphorylation sites clustered around the GTP-binding site (15), suggesting that it may have a functional role that involves its phosphorylation. In contrast to the depression of DAP3, ethanol feeding caused an induction of MRPL12 (Fig.…”

Structure and Function of the Mitochondrial Ribosome

“…Because 28S subunits bind GTP with higher affinity than they do 55S monosomes (123,129), nucleotide hydrolysis by mS29 may be coupled to subunit association and the formation of 55S mitoribosomes (63). In addition to its putative regulation by nucleotide binding, mS29 contains several phosphorylation sites (130,131), which are located close to the regions of the protein involved in the formation of intersubunit bridges (Figure 6b) (62). Therefore, mS29 may function in the regulation of mitochondrial translation and mitoribosomal subunit association, depending on its nucleotide state and phosphorylation status.…”