How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3β phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3β is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Background White matter changes (WMC) on CT or MR brain imaging are common in TIA and stroke patients. Inconsistent associations, never so far researched in stroke and TIA population-based cohorts, have been reported between WMC and severity of stroke, disability, risk of recurrent stroke and death. Methods In consecutive TIA and stroke patients, semiquantitatively rated WMC on CT and MRI, were related to premorbid disability (modified Rankin Score-mRS), baseline MMSE, risk of recurrent stroke, disability at 1 year and death during follow-up. Logistic and Cox regression analyses were adjusted for age, sex, and vascular risk factors. Results 1840 patients had MRI (520) and/or CT (1717). WMC were associated with worse premorbid mRS (OR for mRS>2, given WMC mod/severe on ARWMC scale=1.82, 95% CI 1.35 to 2.45, p=<0.0001) and with risk of worsening mRS at 1 year (OR=1.36, 1.02-1.83, p=0.04). Analyses of Barthel index domains showed associations with reduced pre-morbid mobility and continence. WMC were associated with baseline MMSE <24 (OR=1.71, 1.19-2.47, p=0.004) and with higher risk of death during follow-up, particularly in patients aged <75 years (adjusted HR=1.70, 1.08-2.67, p=0.02). No association emerged with recurrence of ischaemic stroke, although WMC did predict intracerebral haemorrhage (adjusted HR=2.83, 1.17-6.85, p=0.02). Conclusions In TIA or stroke patients, WMC are associated with pre-morbid disability and baseline cognitive impairment and predict progression of disability and risk of death at follow-up, independent of age, sex and vascular risk factors.