Treatment of mammalian cells with the immunosuppressant rapamycin, a bacterial macrolide, selectively suppresses mitogen‐induced translation of an essential class of mRNAs which contain an oligopyrimidine tract at their transcriptional start (5′TOP), most notably mRNAs encoding ribosomal proteins and elongation factors. In parallel, rapamycin blocks mitogen‐induced p70 ribosomal protein S6 kinase (p70s6k) phosphorylation and activation. Utilizing chimeric mRNA constructs containing either a wild‐type or disrupted 5′TOP, we demonstrate that an intact polypyrimidine tract is required for rapamycin to elicit an inhibitory effect on the translation of these transcripts. In turn, a dominant‐interfering p70s6k, which selectively prevents p70s6k activation by blocking phosphorylation of the rapamycin‐sensitive sites, suppresses the translation of the chimeric mRNA containing the wild‐type but not the disrupted 5′TOP. Conversion of the principal rapamycin‐sensitive p70s6k phosphorylation site, T389, to an acidic residue confers rapamycin resistance on the kinase and negates the inhibitory effects of the macrolide on 5′TOP mRNA translation in cells expressing this mutant. The results demonstrate that the rapamycin block of mitogen‐induced 5′TOP mRNA translation is mediated through inhibition of p70s6k activation.

The immunosuppressant rapamycin blocks p7Os"6/p85s activation and phosphorylation of 40S ribosomal protein S6 in Swiss 3T3 cells. (it) rapamycin may inhibit cell growth by blocking S6 phosphorylation and, thus, translation of these mRNAs.The immunosuppressive macrolide rapamycin either reduces or abolishes the rate at which cells enter S phase and subsequent proliferation, depending on the cell type (1). This effect is elicited through the association of rapamycin with an intracellular FK506-and rapamycin-binding protein, termed FKBP (1, 2). Recently, rapamycin was shown to block the mitogen-induced activation of p7os6k/p85s6k and to rapidly inactivate the kinase in mitogen-stimulated cells (3-6). This inhibition is apparently not exerted on the kinase but is exerted on a component involved in controlling its activity (3)(4)(5)(6). The effect of rapamycin appears selective, as the macrolide does not block the activation of other kinasessuch as p74raf, p42mapk, or p--Iskthat are also triggered to act within minutes of mitogen addition (3-6). Activation of p70s6k/p85s6k itself has been associated with the phosphorylation offour residues that exhibit Ser/Thr-Pro motifs and are located within 14 residues of one another in a putative autoinhibitory domain (7). However, rapamycin does not abolish p70s6k/p85s6k activity through altering the phosphorylated state of these sites but works through a specific set of sites, which apparently turn over slowly or not at all (8).The p70s6k/p85s6k represent two isoforms of the same enzyme, derived by differential splicing from a common gene (9) and whose target is 40S ribosomal protein S6 (10). The p85s6k sequence is identical to that of p70s6k, except for a 23-aa extension at its amino terminus, which contains a nuclear-targeting sequence (9), consistent with the finding of S6 phosphorylation in the nucleus after mitogenic stimulation (11). The five phosphorylation sites within S6 reside in a 15-aa fragment at the carboxyl terminus and are phosphorylated in a specific order (10). The kinase is highly selective for S6, exhibiting a Km of 0.25 u.M (12), and in vitro phosphorylates four and, possibly, the fifth site observed in vivo (13). By using several approaches, S6 has been mapped to the tRNAmRNA-binding site of the 40S ribosome (14) and in the phosphorylated state has been implicated in the activation of protein synthesis, as well as in alterations in the pattern of translation (10). Because rapamycin blocks S6 phosphorylation, presumably by inhibiting p70s6k/p85s6k (3), its inhibitory effects on cell growth may be through inhibiting S6 phosphorylation and thus, protein synthesis.We examined the effect of rapamycin on p70s6k/p85s6k activity and S6 phosphorylation in quiescent and serumstimulated cells. Then we analyzed the role of the macrolide in the activation of specific mRNA transcripts. Our results show that rapamycin has an inhibitory effect on the activation of protein synthesis and, more importantly, that this effect is through suppressing translation of a fam...

Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy, which is positively regulated by the JNK/c-Jun pathway and is defective in the injured central nervous system.

Phosphoinositides have crucial roles in cellular controls, many of which have been established through the use of small-molecule inhibitors. Here, we describe YM201636, a potent inhibitor of the mammalian class III phosphatidylinositol phosphate kinase PIKfyve, which synthesizes phosphatidylinositol 3,5-bisphosphate. Acute treatment of cells with YM201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. Inhibitor specificity is shown by the use of short interfering RNA against the target, as well as by rescue with the drug-resistant yeast orthologue Fab1. We concluded that the phosphatidylinositol 3,5-bisphosphate pathway is integral to endosome formation, determining morphology and cargo flux.

Activation of the Raf/MAP kinase pathway is a critical event in tumorigenesis induced by RAS and other oncogenes, a major role of this signaling system being the regulation of cellular transcription factors. To address the contribution of MAP kinase mediated transcriptional changes to the transformed phenotype, we used an inducible form of Raf to analyze early changes in the transcription of some 6000 genes following activation of the kinase in a normal human breast epithelial cell line. Of the more than 120 significant changes in mRNA level detected, genes promoting cell proliferation, invasiveness, and angiogenesis featured prominently. Some of the most strongly induced genes encoded growth factors of the EGF family: Autocrine activation of the EGF receptor was shown to be responsible for the ability of Raf activation to protect these cells from apoptosis induced by detachment of cells from extracellular matrix (anoikis), which is a critical component of the transformed phenotype.

ATG9A is a multispanning membrane protein essential for autophagy. Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation. ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome. To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid–starved cells was performed. These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes. Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site. PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response. PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes.

SummaryAutophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.

SummaryStarvation-induced autophagy requires activation of the ULK complex at the phagophore. Two Golgi proteins, WAC and GM130, regulate autophagy, however their mechanism of regulation is unknown. In search of novel interaction partners of WAC, we found that GM130 directly interacts with WAC, and this interaction is required for autophagy. WAC is bound to the Golgi by GM130. WAC and GM130 interact with the Atg8 homolog GABARAP and regulate its subcellular localization. GABARAP is on the pericentriolar matrix, and this dynamic pool contributes to autophagosome formation. Tethering of GABARAP to the Golgi by GM130 inhibits autophagy, demonstrating an unexpected role for a golgin. WAC suppresses GM130 binding to GABARAP, regulating starvation-induced centrosomal GABARAP delivery to the phagophore. GABARAP, unlipidated and lipidated, but not LC3B, GABARAPL1, and GATE-16, specifically promotes ULK kinase activation dependent on the ULK1 LIR motif, elucidating a unique non-hierarchical role for GABARAP in starvation-induced activation of autophagy.