Mitochondria are highly dynamic organelles that mediate essential cell functions such as apoptosis and cell-cycle control in addition to their role as efficient ATP generators. Mitochondrial morphology changes are tightly regulated, and their shape can shift between small, fragmented units and larger networks of elongated mitochondria. We demonstrate that mitochondrial elements become significantly elongated and interconnected shortly after nutrient depletion. This mitochondrial morphological shift depends on the type of starvation, with an additive effect observed when multiple nutrients are depleted simultaneously. We further show that starvation-induced mitochondrial elongation is mediated by downregulation of dynamin-related protein 1 (Drp1) through modulation of two Drp1 phosphorylation sites, leading to unopposed mitochondrial fusion. Finally, we establish that mitochondrial tubulation upon nutrient deprivation protects mitochondria from autophagosomal degradation, which could permit mitochondria to maximize energy production and supply autophagosomal membranes during starvation.autophagy | mitofusin M itochondria are dynamic organelles that mediate many essential cell functions. Depending on the cellular context, mitochondria shift between fragmented and tubular network-like morphologies by means of coordinated fission and fusion (1, 2). Proteins responsible for mitochondrial fusion include the outer mitochondrial membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) (3-5) and the inner membrane protein optic atrophy 1 (Opa1) (6). Fission is mediated by dynamin-related protein 1 (Drp1) (7,8) and its interaction with binding partners Fis1 and/or mitochondrial fission factor (Mff) (9, 10). Multiple mechanisms, including phosphorylation, sumoylation, and ubiquitination, coordinate Drp1 fission capacity (11)(12)(13)(14). Phosphorylation of Drp1 at S616 by Cdk1/cyclin B results in increased Drp1 fission activity (13). Conversely, phosphorylation of Drp1 at S637 by PKA decreases fission by causing Drp1 retention in the cytosol, whereas dephosphorylation of S637 by calcineurin causes Drp1 translocation to the mitochondria and increased mitochondrial fission (11, 15).Mitochondrial morphological dynamics are linked to regulation of many specific cell functions. Changes in mitochondrial cristae and mitochondrial fragmentation, for example, play a vital role in apoptosis (16,17). Ca 2+ transfer (18), cell-cycle regulation (13,19), and mitochondrial quality control (20, 21) are all closely tied to changes in mitochondrial morphology. Furthermore, stress conditions and changes in energy source can induce significant mitochondrial morphological changes (22, 23). Very recently, nutrient starvation was shown to induce mitochondrial elongation and to protect mitochondria from autophagic degradation (24). In addition to the above functions, mitochondria have recently been linked to autophagosome biogenesis during starvation conditions (25), and it is possible that mitochondrial morphological changes play a role in th...

The protein kinase C (PKC) Ser/Thr kinases account for approximately 2% of the human kinome and regulate diverse cellular behaviors. PKC catalytic activity requires priming phosphorylations at three conserved sites within the kinase domain. Here we demonstrate that priming of PKC is dependent on the conformation of the nucleotide binding pocket but not on its intrinsic kinase activity. Inactive ATP binding site mutants are unprimed, but they become phosphorylated upon occupancy of the ATP binding pocket with inhibitors of PKC. We have exploited this property to screen for PKC inhibitors in vivo. Further, we generated a distinct class of kinase-inactive mutants that maintain the integrity of the ATP binding pocket; such mutants are constitutively primed and functionally distinct from ATP binding site mutants. These data demonstrate that autophosphorylation is not required for PKC priming and show how ATP pocket occupation can enable a kinase to mature as well as function.

The phosphoserine/threonine binding protein 14‐3‐3 stimulates the catalytic activity of protein kinase C‐ε (PKCε) by engaging two tandem phosphoserine‐containing motifs located between the PKCε regulatory and catalytic domains (V3 region). Interaction between 14‐3‐3 and this region of PKCε is essential for the completion of cytokinesis. Here, we report the crystal structure of 14‐3‐3ζ bound to a synthetic diphosphorylated PKCε V3 region revealing how a consensus 14‐3‐3 site and a divergent 14‐3‐3 site cooperate to bind to 14‐3‐3 and so activate PKCε. Thermodynamic data show a markedly enhanced binding affinity for two‐site phosphopeptides over single‐site 14‐3‐3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14‐3‐3 ligand. This dual‐site intra‐chain recognition has implications for other 14‐3‐3 targets, which seem to have only a single 14‐3‐3 motif, as other lower affinity and cryptic 14‐3‐3 gatekeeper sites might exist.

Escherichia coli ZiPD is the best characterized protein encoded by the elaC gene family and is a model for the 3-pre-tRNA processing endoribonucleases (tRNase Z). A metal ligand-based sequence alignment of ZiPD with metallo-␤-lactamase domain proteins of known crystallographic structure identifies a ZiPD-specific sequence insertion of ϳ50 residues, which we will refer to as the ZiPD exosite. Functionally characterized ZiPD homologs from Bacillus subtilis, Methanococcus janaschii, and human share the presence of the ZiPD exosite, which is also present in the amino-terminal, but not in the carboxyl-terminal, domain of ElaC2 proteins. Another class of functionally characterized tRNase Z enzymes from Thermotoga maritima and Arabidopsis thaliana lack characteristic motifs in the exosite but possess a sequence segment with clustered basic amino acid residues. As an experimental attempt to investigate the function of the exosite we constructed a ZiPD variant that lacks this module (ZiPD⌬). ZiPD⌬ has almost wild-type-like catalytic properties for hydrolysis of the small, chromogenic substrate bis(p-nitrophenyl)phosphate. Removal of the ZiPD exosite only affects k cat, which is reduced by less than 40%, whereas both K and the Hill coefficient (measures of the substrate affinity and cooperativity, respectively) remain unchanged. Hence, the exosite is not required for the intrinsic phosphodiesterase activity of ZiPD. Removal of the exosite also does not affect the dimerization properties of ZiPD. In contrast to the wild-type enzyme, ZiPD⌬ does not process pre-tRNA, and gel shift assays demonstrate that only the wild-type enzyme, but not ZiPD⌬, binds mature tRNA. These findings show that the exosite is essential for pre-tRNA recognition. In conclusion, we identify a ZiPD exosite that guides physiological substrate recognition in the ZiPD/ElaC protein family.

SummaryAtypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.

The elaC gene product from Escherichia coli, ZiPD, is a 3 tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the elaC homologue from Bacillus subtilis, E. coli elaC is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3 end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in E. coli are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 Å. This structure shares many features with the tRNase Z enzymes from B. subtilis and Thermotoga maritima, but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the B. subtilis enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.The precise cleavage of precursor tRNA (pre-tRNA) after its transcription at both the 5Ј and 3Ј extensions by specific nucleases is a vital step in the maturation of tRNA (18). tRNase Z, a member of the metallo-␤-lactamase superfamily, has been identified as an important endonuclease which cleaves the 3Ј extension from various tRNA precursors (24). tRNase Z enzymes have been functionally characterized from a number of both bacterial and archaeal organisms, as well as eukaryotes, including humans (31).In most cases, tRNase Z proteins cleave the 3Ј extension solely from pre-tRNA molecules that lack the CCA triplet. This CCA triplet is the sequence that is present at the 3Ј end of all mature tRNA molecules (30), and it is vital for association of tRNA with the large ribosomal subunit during translation (10,19). While some tRNAs undergo 3Ј cleavage followed by addition of the CCA triplet, some archaeal and bacterial tRNAs contain an encoded CCA triplet, and the majority of these tRNAs require neither cleavage by tRNase Z nor subsequent CCA addition. With the exception of Thermotoga maritima, all tRNase Z enzymes characterized in this respect to date cleave only pre-tRNA lacking the CCA triplet in vivo (17, 25).The Escherichia coli elaC gene product, designated ZiPD (34), ecoZ (24), tRNase Z (17), or RNase BN (9), is unique in this respect because although all E. coli tRNAs are transcribed with an encoded CCA triplet, recombinant E. coli ZiPD has been shown to be capable of binding and cleaving only exogenous pre-tRNA which does not contain a CCA triplet (17,27,31). Although limited tRNase Z activity has been found in vivo (13, 16), after deletion of the elaC gene in E. coli ...

The PKB (protein kinase B) and PKC (protein kinase C) families display highly related catalytic domains that require a largely conserved series of phosphorylations for the expression of their optimum activities. However, in cells, the dynamics of these modifications are quite distinct. Based on experimental evidence, it is argued that the underlying mechanisms determining these divergent behaviours relate to the very different manner in which their variant regulatory domains interact with their respective catalytic domains. It is concluded that the distinct behaviours of PKB and PKC proteins are defined by the typical ground states of these proteins.

Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.