No abstract

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser͞Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G 1 and G2͞M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5͞eIF2͞eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation. C asein kinase 2 (CK2) (1-4) is composed of two subunits, ␣ or ␣Ј and ␤, which combine to form a native ␣ 2 ␤ 2 tetramer. Disruption of the catalytic subunits (␣ and ␣Ј) is lethal in Saccharomyces cerevisiae (5) and disruption of the regulatory ␤ subunit in mice leads to early embryonic lethality (6). CK2 phosphorylates a range of cellular targets in a variety of subcellular sites and appears to be highly pleiotropic; it is involved in many key biological functions, including growth and cell cycle control (7), signal transduction (3), circadian rhythms (8, 9), and gene expression (10, 11). CK2 is also a stress-activated kinase and might participate in the transduction of survival signals to avoid damage by mutagenic UV radiation (12, 13). An important role for CK2 in promoting cell proliferation and transformation has been indicated by several studies. In mammalian systems, its targeted overexpression in mice results in the development of T cell lymphoma and mammary tumorigenesis (5). Despite these findings, there is still much uncertainty regarding the activation of CK2 in response to stimuli (14). The mechanism by which it is regulated and its precise function in cell cycle progression and proliferation is still poorly understood.CK2 activity and stability are believed to be regulated in part by holoenzyme formation via a self-assembly mechanism and by phosphorylation. Phosphorylation by p34 cdc2 of the catalytic ␣ subunit at the C-terminal domain occurs in ...