| As a major part of cellular compartmentalization,proteins achieve specific subcellular localization through sorting or post-translational modifications.Elucidating the subcellular localization of the proteome is of great significance for analyzing functions and regulations of proteins,which are underlying signal transduction networks governing various cellular progresses.Phosphorylation is one of the most wide-spread and well-studied post-translational modifications of proteins.It plays regulatory roles in most cellular progresses,such as metabolism,proliferation,differentiation,and motility.Protein kinases are enzymes that catalyze protein phosphorylation in cells.The human genome encodes 538 protein kinases,which are collectively referred as the kinome.They account for about 1.7% of genes encoded by the genome.In addition to important physiological functions,dysregulation of protein kinases plays important roles in human diseases,especially cancer.They have also become the most explored targets in precisional medicine.Although many studies have investigated subcellular localizations,functions and regulatory mechanisms of specific protein kinases,complete characterization of subcellular localizations of the kinome is still lacking.In this study,we constructed a plasmid library comprising more than 85% of the kinome.We then mapped the subcellular localization of each kinase by immunofluorescence staining in transfected HeLa cells under a unified condition.This collection of information is summarized in the Kinome Atlas(KA).As shown by KA,even cytoplasm and nucleus were main localizations,many kinases are organelle-specific.By alignment with protein localization databases,we found many protein kinases with previously unknown specific subcellular localization.Mitochondrion is a multifunctional organelle playing important roles such as energy metabolism and apoptosis regulation.We therefore focused on mitochondria-localized kinases.By comparing with a database of mitochondrial proteome,we found that most of the known mitochondrial protein kinases still exhibited clear mitochondrial localization in KA,confirming the reliability of our method.Furthermore,we discovered a number of kinases with previously unknown mitochondrial localizations.By super-resolution microscopy and other methods,we determined sub-mitochondrial localizations of these kinases.We next focused on a mitochondrial intermembrane space-localized kinase MOK.By mutational analysis we found that the mitochondrial localization of MOK dependents on two regions in its kinase domain and C-terminal domain,respectively.Although MOK lacks classical mitochondrial localization signal,proteins of the mitochondrial transporter complexes such as TOM20 and TIM23 are still necessary for the mitochondrial localization of MOK.Knockout of MOK in A375 cells resulted in a series of changes,including decreasing of mitochondria cristae,repression of mitochondrial respiration,decreasing of cellular ATP level,and enhanced ROS and cell death under oxidative stress.These findings demonstrate that MOK plays an important role in mitochondrial functions.Unexpectedly,MOK2,a splicing isoform with better mitochondrial localization but defective kinase activity could rescue functional defects caused by MOK knockout.This finding suggests that MOK regulates mitochondrial functions independ of its kinase activity.By tandem affinity purification,we identified ATPase family AAA domain containing 3A(ATAD3A)as a binding protein of MOK.Knockout of ATAD3 A also resulted in a decrease of mitochondrial cristae.Therefore,it is possible that MOK regulates the structure of mitochondria through ATAD3 A.Our study systematically characterized subcellular localizations of the human kinome,and further elucidated the localization mechanism and function of a mitochondria-localized kinase MOK.This study laid a foundation for further investigations of novel mitochondrial kinases and kinome functions in previously unknown subcellular localizations. |
