APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve complex

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer’s disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein–protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer’s disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer’s disease

Functional interaction between RAFT1/FRAP/mTOR and protein kinase Cδ in the regulation of cap-dependent initiation of translation

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Here we demonstrate that protein kinase Cδ (PKCδ) associates with RAFT1 and that PKCδ is required for the phosphorylation and inactivation of 4E-BP1. PKCδ-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCδ inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCδ inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCδ associates with RAFT1 and thereby regulates phosphorylation of 4E–BP1 and cap-dependent initiation of protein translation

Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction

Impaired ribosome biogenesis is attributed to nucleolar disruption and diffusion of a subset of 60S ribosomal proteins, particularly ribosomal protein (rp)L11, into the nucleoplasm, where they inhibit MDM2, leading to p53 induction and cell-cycle arrest(1–4). Previously, we demonstrated that deletion of the 40S rpS6 gene in mouse liver prevents hepatocytes from re-entering the cell cycle after partial hepatectomy(5). Here, we show that this response leads to an increase in p53, which is recapitulated in culture by rpS6-siRNA treatment and rescued by the simultaneous depletion of p53. However, disruption of biogenesis of 40S ribosomes had no effect on nucleolar integrity, although p53 induction was mediated by rpL11, leading to the finding that the cell selectively upregulates the translation of mRNAs with a polypyrimidine tract at their 5´-transcriptional start site (5´-TOP mRNAs), including that encoding rpL11, on impairment of 40S ribosome biogenesis. Increased 5´-TOP mRNA translation takes place despite continued 60S ribosome biogenesis and a decrease in global translation. Thus, in proliferative human disorders involving hypomorphic mutations in 40S ribosomal proteins(6,7), specific targeting of rpL11 upregulation would spare other stress pathways that mediate the potential benefits of p53 induction(8)