ABSTRACT
TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.
Subject(s)
14-3-3 Proteins/genetics , Fetal Proteins/genetics , Lymphocytes/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Protein-Tyrosine Kinases/genetics , Ubiquitin/genetics , 14-3-3 Proteins/metabolism , A549 Cells , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Fetal Proteins/antagonists & inhibitors , Fetal Proteins/metabolism , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Lymphocytes/drug effects , Lymphocytes/pathology , Mice , Phospholipase C gamma/genetics , Phospholipase C gamma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Pyrimidines/pharmacology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Signal Transduction , Survival Analysis , Tumor Burden/drug effects , Ubiquitin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival.
Subject(s)
Acylation/physiology , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mutation/genetics , Superoxide Dismutase-1/metabolism , Superoxide Dismutase/metabolism , Acylation/genetics , Amyotrophic Lateral Sclerosis/genetics , Animals , Humans , Mice , Mitochondria/genetics , Oxidative Stress/physiology , Superoxide Dismutase/genetics , Superoxide Dismutase-1/chemistry , Superoxide Dismutase-1/geneticsABSTRACT
The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, Lys(49) and Lys(120), is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the Lys(49)/Lys(120) deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well described interacting partners, Bad and AS160, which triggers their dephosphorylation at Ser(112) and Thr(642), respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and Ser(112)/Thr(642) phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.
