Fyn kinase regulates translation in mammalian mitochondria

Highlights

• Fyn kinase is associated with mitochondrial translation machinery.

• Mammalian mitochondrial ribosomal proteins are Tyr-phosphorylated by Fyn kinase in vitro.

• Fyn regulates protein synthesis and oxidative phosphorylation in mammalian mitochondria.

Abstract

Background

Mitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known.

Methods

Mitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches.

Results

Here, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes.

Conclusions and general significance

Our findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria.

Introduction

Mitochondrial translation is responsible for the synthesis of 13 mitochondrially-encoded subunits of oxidative phosphorylation (OXPHOS) complexes, which generate approximately 90% of the energy for a eukaryotic cell in the form of ATP. In addition to the changes and defects in expression of mitochondrial ribosomal proteins (MRPs) and translation factors, post-translational modifications (PTMs) of these translation components are important for the regulation of OXPHOS in mammalian mitochondria [1], [2], [3], [4]. Our laboratory has previously shown that MRPs are modified by phosphorylation and acetylation to regulate the synthesis of these 13 essential proteins [1], [4]. In the phosphoproteomic analysis of bovine 55S ribosomes, we identified at least twenty-four phosphorylated MRPs at Ser/Thr or Tyr residues [1], [2]. Since the initial discovery of phosphorylation of MRPs, high throughput analyses of phosphoproteomes of normal and diseased tissues and cell lines revealed that the majority of MRPs were phosphorylated [5], [6], [7], [8], [9]. Interestingly, the phosphorylation sites in these proteins are mostly located in functionally significant regions of the ribosome, possibly involved in regulation of the function of these sites in protein synthesis and energy metabolism in health and disease [2], [10]. In fact, phosphorylation of MRPS29, also known as DAP3, is critical for its role in induction of apoptosis in various cell lines [2], [11]. Changes in the expression and phosphorylation of DAP3 in various cancer cells and tumors suggest a significant role for this modification in disease states [11], [12], [13]. However, the investigation of the role of a specific phosphorylation instance in MRPs is a challenging task because they are part of a large ribonucleoprotein complex.

One of the current approaches to study dynamic changes of PTMs and their role in regulation of multi-subunit complexes such as ribosomes is to identify the enzyme(s) responsible for these PTMs. With this approach, we have identified the ribosome-associated SIRT3 as the NAD⁺-dependent deacetylase responsible for the deacetylation of MRPL10 and shown its role in protein synthesis [4]. Similarly, several Ser/Thr kinases and members of the Src family tyrosine kinases (SFKs) have emerged as major regulators of energy metabolism in mammalian mitochondria in health and disease [14], [15]. Although localization of Ser/Thr kinases into the mitochondria is not clear, SFK members such as cSrc and Fyn were shown to translocate into the mitochondria and phosphorylate subunits of complex I, II, and IV and pyruvate dehydrogenase in mammals [16], [17], [18], [19]. In a recent study, Matsushima et al. reported that Fyn kinase and NOX4 are co-located in mitochondria and regulate ROS production and cell death in cardiomyocytes [20]. Specifically, the Tyr phosphorylation of pyruvate dehydrogenase by SFKs was shown to be one of the key regulators of this complex in promoting the Warburg effect in various cancers [19]. These observations are also supported by the inhibition of the OXPHOS process in osteosarcoma and prostate cancer cell lines treated with SFK inhibitors [21], [22].

In this study, we provide the first evidence for a kinase responsible for phosphorylation of MRPs and 55S ribosomes in vitro. Fractionation by strong cation exchange (SCX) chromatography of highly purified mitoplasts has resulted in a fraction with high kinase activity. Protein identification in this fraction by LC-MS/MS revealed the presence of Fyn kinase associated with this high kinase activity. Furthermore, the detection of MRPs and translation factors in the same fraction supported the role of this kinase in the regulation of mitochondrial translation by phosphorylation. siRNA-directed knock-down of Fyn kinase in human cell lines reduced the expression of a mitochondrially encoded subunit of complex IV, while over-expression stimulated the synthesis of this subunit. These observations led us to propose that the Fyn-dependent phosphorylation of mitochondrial translation components is involved in regulation of OXPHOS and energy metabolism in mammalian mitochondria.