Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p

Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the αA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.     

The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p.

The Saccharomyces cerevisiae Sln1 protein is a ''two-component'' regulator involved in osmotolerance. Two-component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.     

Phosphorylated Ssk1 prevents unphosphorylated Ssk1 from activating the Ssk2 mitogen-activated protein kinase kinase kinase in the yeast high-osmolarity glycerol osmoregulatory pathway

In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1~P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1~P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.     

Regulation of ubiquitin-proteasome system mediated degradation by cytosolic stress

ER-associated, ubiquitin-proteasome system (UPS)-mediated degradation of the wild-type (WT) gap junction protein connexin32 (Cx32) is inhibited by mild forms of cytosolic stress at a step before its dislocation into the cytosol. We show that the same conditions (a 30-min, 42 degrees C heat shock or oxidative stress induced by arsenite) also reduce the endoplasmic reticulum (ER)-associated turnover of disease-causing mutants of Cx32 and the cystic fibrosis transmembrane conductance regulator (CFTR), as well as that of WT CFTR and unassembled Ig light chain. Stress-stabilized WT Cx32 and CFTR, but not the mutant/unassembled proteins examined, could traverse the secretory pathway. Heat shock also slowed the otherwise rapid UPS-mediated turnover of the cytosolic proteins myoD and GFPu, but not the degradation of an ubiquitination-independent construct (GFP-ODC) closely related to the latter. Analysis of mutant Cx32 from cells exposed to proteasome inhibitors and/or cytosolic stress indicated that stress reduces degradation at the level of substrate polyubiquitination. These findings reveal a new link between the cytosolic stress-induced heat shock response, ER-associated degradation, and polyubiquitination. Stress-denatured proteins may titer a limiting component of the ubiquitination machinery away from pre-existing UPS substrates, thereby sparing the latter from degradation.     

Cigarette smoke induces Akt protein degradation by the ubiquitin-proteasome system

Emphysema is one of the characteristic features of chronic obstructive pulmonary disease, which is caused mainly by cigarette smoking. Recent data have suggested that apoptosis and cell cycle arrest may contribute to the development of emphysema. In this study, we addressed the question of whether and how cigarette smoke affected Akt, which plays a critical role in cell survival and proliferation. In normal human lung fibroblasts, cigarette smoke extract (CSE) caused cell death, accompanying degradation of total and phosphorylated Akt (p-Akt), which was inhibited by MG132. CSE exposure resulted in preferential ubiquitination of the active Akt (myristoylated), rather than the inactive (T308A/S473A double mutant) Akt. Consistent with cytotoxicity, CSE induced a progressive decrease of phosphorylated human homolog of mouse double minute homolog 2 (p-HDM2) and phosphorylated apoptosis signal regulating kinase 1 (p-ASK1) with concomitant elevation of p53, p21, and phosphorylated p38 MAPK. Forced expression of the active Akt reduced both CSE-induced cytotoxicity and alteration in HDM2/p53/p21 and ASK1/p38 MAPK, compared with the inactive Akt. Of note, CSE induced expression of the tetratrico-peptide repeat domain 3 (TTC3), known as a ubiquitin ligase for active Akt. TTC3 siRNAs suppressed not only CSE-induced Akt degradation but also CSE-induced cytotoxicity. Accordingly, rat lungs exposed to cigarette smoke for 3 months showed elevated TTC3 expression and reduced Akt and p-Akt. Taken together, these data suggest that cigarette smoke induces cytotoxicity, partly through Akt degradation via the ubiquitin-proteasome system, in which TTC3 acts as a ubiquitin ligase for active Akt.     

Ursodeoxycholic acid modulates the ubiquitin-proteasome degradation pathway of p53

p53/Mdm-2 interaction is a prime target of ursodeoxycholic acid (UDCA) for regulating apoptosis in primary rat hepatocytes. Here, we further explored the role of UDCA in downregulating p53 by Mdm-2. UDCA reduced the stability of p53 by decreasing protein half-life. Although proteasomal activity was slightly increased with UDCA, the effect was also observed for other bile acids. More importantly, immunoprecipitation assays revealed that UDCA promoted p53 ubiquitination, therefore leading to increased p53 degradation. In this regard, proteasome inhibition after UDCA pre-treatment resulted in accumulation of ubiquitinated p53, which in turn was prevented in cells overexpressing a mutated form of p53 that does not undergo Mdm-2 ubiquitination. The involvement of Mdm-2 in UDCA-mediated response was further confirmed by siRNA-mediated gene silencing experiments. Finally, the protective effect of UDCA against p53-induced apoptosis was abolished after inhibition of proteasome activity and prevention of p53 ubiquitination by Mdm-2. These findings suggest that UDCA protects cells from p53-mediated apoptosis by promoting its degradation via the Mdm-2-ubiquitin-proteasome pathway.     

The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway

The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.     

Diversity of degradation signals in the ubiquitin-proteasome system

The ubiquitin-proteasome system degrades an enormous variety of proteins that contain specific degradation signals, or 'degrons'. Besides the degradation of regulatory proteins, almost every protein suffers from sporadic biosynthetic errors or misfolding. Such aberrant proteins can be recognized and rapidly degraded by cells. Structural and functional data on a handful of degrons allow several generalizations regarding their mechanism of action. We focus on different strategies of degron recognition by the ubiquitin system, and contrast regulatory degrons that are subject to signalling-dependent modification with those that are controlled by protein folding or assembly, as frequently occurs during protein quality control.     

tRNA import into yeast mitochondria is regulated by the ubiquitin-proteasome system

In Saccharomyces cerevisiae, one of two cytosolic lysine-tRNAs is partially imported into mitochondria. We demonstrate that three components of the ubiquitin/26S proteasome system (UPS), Rpn13p, Rpn8p and Doa1p interact with the imported tRNA and with the essential factor of its mitochondrial targeting, pre-Msk1p. Genetic and biochemical assays demonstrate that UPS plays a dual regulatory role, since the overall inhibition of cellular proteasome activity reduces tRNA import, while specific depletion of Rpn13p or Doa1p increases it. This result suggests a functional link between UPS and tRNA mitochondrial import in yeast and indicates on the existence of negative and positive import regulators.     

Contributions of the response regulators Ssk1p and Skn7p in the pseudohyphal development, stress adaptation, and drug sensitivity of the opportunistic yeast Candida lusitaniae

We recently characterized the histidine kinase receptor genes of Candida lusitaniae. For the present study, we have further investigated the role of SSK1 and SKN7, encoding response regulators. The results of functional analysis of mutants indicated that Ssk1p is involved in osmotolerance and pseudohyphal development, whereas Skn7p appears crucial for oxidative stress adaptation.     

Requirement of dendritic Akt degradation by the ubiquitin-proteasome system for neuronal polarity

Asymmetric distributions of activities of the protein kinases Akt and glycogen synthase kinase 3beta (GSK-3beta) are critical for the formation of neuronal polarity. However, the mechanisms underlying polarized regulation of this pathway remain unclear. In this study, we report that the instability of Akt regulated by the ubiquitin-proteasome system (UPS) is required for neuron polarity. Preferential distribution in the axons was observed for Akt but not for its target GSK-3beta. A photoactivatable GFP fused to Akt revealed the preferential instability of Akt in dendrites. Akt but not p110 or GSK-3beta was ubiquitinated. Suppressing the UPS led to the symmetric distribution of Akt and the formation of multiple axons. These results indicate that local protein degradation mediated by the UPS is important in determining neuronal polarity.     

Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family

The AAA protein p97 is a central component in the ubiquitin-proteasome system, in which it is thought to act as a molecular chaperone, guiding protein substrates to the 26S proteasome for degradation. This function is dependent on association with cofactors that are specific to the different biological pathways p97 participates in. The UBX-protein family (ubiquitin regulatory X) constitutes the largest known group of p97 cofactors. We propose that the regulation of p97 by UBX-proteins utilizes conserved structural features of this family. Firstly, they act as scaffolding subunits in p97-containing multiprotein complexes, by providing additional interaction motifs. Secondly, they provide regulation of multiprotein complex assembly and we suggest two possible models for p97 substrate recruitment in the UPS pathway. Lastly, they impose constraints on p97 and its interaction with substrates and further cofactors. These features allow the regulation, within the UPS, of the competitive interactions on p97, a regulation that is crucial to allow the diverse functionality of p97.     

Insight into the role of HOG pathway components Ssk2p, Pbs2p, and Hog1p in the opportunistic yeast Candida lusitaniae

In the present study, we have investigated the role of SSK2, PBS2, and HOG1, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in Candida lusitaniae. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.