Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3.
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 Cdelta 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.
Miller
Jennifer L
JL
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Koc
Hasan
H
Koc
Emine C
EC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry
D051017:Apoptosis Regulatory Proteins; C097041:DAP3 protein, human; D050505:Mutant Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D051745:Protein Kinase C-delta; D020169:Caspases