T cell activation requires the import of NF-AT transcription factors to the nucleus, a process promoted by calcineurin-dependent dephosphorylation and inhibited by poorly understood protein kinases. Here, we report the identification of two protein kinases that oppose NF-AT4 nuclear import. Casein kinase Ialpha directly binds and phosphorylates NF-AT4, resulting in the inhibiton of NF-AT4 nuclear translocation. MEKK1 indirectly suppresses NF-AT4 nuclear import by stabilizing the interaction between NF-AT4 and CKIalpha. CKIalpha thus acts to establish an intramolecular masking of the nuclear location signal on NF-AT4, while MEKK1 augments this mechanism, and may further provide a link to signal transduction pathways regulating NF-AT4.

Nuclear import of the NF-AT transcription factors during T-cell activation requires the calcium-activated phosphatase calcineurin, which unmasks nuclear-location signals on NF-AT. We show here that the nuclear import of NF-ATs is not sufficient to activate NF-AT target genes, as NF-ATs are subject to a futile cycling across the nuclear envelope owing to engagement with the exportin protein Crm1. Calcineurin suppresses this futile cycling by a non-catalytic mechanism involving the masking of nuclear export signals on NF-AT targeted by Crm1. This clustering of binding sites for calcineurin and Crml on NF-AT establishes an inherent competition between these molecules that imparts exquisite calcium sensitivity to the shuttling dynamics of the NF-AT transcription factors. Such a balance between nuclear import and export may regulate the action of other transcription factors.

Cell-cycle checkpoints help to protect the genomes of proliferating cells under genotoxic stress. In multicellular organisms, cell proliferation is often directed toward differentiation during development and throughout adult homeostasis. To prevent the formation of differentiated cells with genetic instability, we hypothesized that genotoxic stress may trigger a differentiation checkpoint. Here we show that exposure to genotoxic agents causes a reversible inhibition of myogenic differentiation. Muscle-specific gene expression is suppressed by DNA-damaging agents if applied prior to differentiation induction but not after the differentiation program is established. The myogenic determination factor, MyoD (encoded by Myod1), is a target of the differentiation checkpoint in myoblasts. The inhibition of MyoD by DNA damage requires a functional c-Abl tyrosine kinase (encoded by Abl1), but occurs in cells deficient for p53 (transformation-related protein 53, encoded by Trp53) or c-Jun (encoded by the oncogene Jun). These results support the idea that genotoxic stress can regulate differentiation, and identify a new biological function for DNA damage-activated signaling network.

No abstract

The nuclear factors of activated T cells (NF-ATs) constitute a family of transcription factors that transduce calcium signals in the immune, cardiac, muscular and nervous systems. Like their distant relatives of the Rel family, including NF-kappaB, NF-ATs are cytoplasmic in resting cells and activated by means of induced nuclear import. Unlike NF-kappaB, however, NF-ATs show highly dynamic nuclear shuttling properties that have important implications for graded signaling by these molecules. This review focuses on recent advances in deciphering mechanisms by which calcium signaling regulates the nucleo-cytoplasmic shuttling,and therefore transactivation functions of the NF-ATs. These discoveries highlight the interplay between nuclear import and export signals on NF-ATs, and the roles of the calcium-activated phosphatase calcineurin and NF-AT kinases in controlling the activity of these signals. They also reveal that NF-ATs, as well as other transcription factors controlled at the level of nuclear import, face the very real prospect of futile cycling across the nuclear envelope as a consequence of conflicting nuclear import and export signals. We discuss the molecular mechanisms by which calcineurin suppresses futile cycling, as well as the major challenges to our understanding of NF-AT signaling in diverse biological systems.