Biochemical Journal - BJ Signal

GRAS proteins: the versatile roles of intrinsically disordered proteins in plant signalling

Xiaolin Sun, William T. Jones and Erik H. A. Rikkerink — 2012-02-15

IDPs (intrinsically disordered proteins) are highly abundant in eukaryotic proteomes and important for cellular functions, especially in cell signalling and transcriptional regulation. An IDR (intrinsically disordered region) within an IDP often undergoes disorder-to-order transitions upon binding to various partners, allowing an IDP to recognize and bind different partners at various binding interfaces. Plant-specific GRAS proteins play critical and diverse roles in plant development and signalling, and act as integrators of signals from multiple plant growth regulatory and environmental inputs. Possessing an intrinsically disordered N-terminal domain, the GRAS proteins constitute the first functionally required unfoldome from the plant kingdom. Furthermore, the N-terminal domains of GRAS proteins contain MoRFs (molecular recognition features), short interaction-prone segments that are located within IDRs and are able to recognize their interacting partners by undergoing disorder-to-order transitions upon binding to these specific partners. These MoRFs represent potential protein–protein binding sites and may be acting as molecular bait in recognition events during plant development. Intrinsic disorder provides GRAS proteins with a degree of binding plasticity that may be linked to their functional versatility. As an overview of structure–function relationships for GRAS proteins, the present review covers the main biological functions of the GRAS family, the IDRs within these proteins and their implications for understanding mode-of-action.

Ubiquitin links to cytoskeletal dynamics, cell adhesion and migration

Antje Schaefer, Micha Nethe and Peter L. Hordijk — 2012-02-15

Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein–protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.

Palmitoylation and trafficking of GAD65 are impaired in a cellular model of Huntington's disease

2012-02-15

HD (Huntington's disease) is caused by an expanded polyQ (polyglutamine) repeat in the htt (huntingtin protein). GABAergic medium spiny neurons in the striatum are mostly affected in HD. However, mhtt (mutant huntingtin)-induced molecular changes in these neurons remain largely unknown. The present study focuses on the effect of mhtt on the subcellular localization of GAD (glutamic acid decarboxylase), the enzyme responsible for synthesizing GABA (γ-aminobutyric acid). We report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus of mhtt or full-length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal htt; however, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of HIP14 (huntingtin-interacting protein 14), the enzyme responsible for palmitoylating GAD65 in vivo, could rescue GAD65 palmitoylation and vesicle-associated GAD65 trafficking. Taken together, our data support the idea that GAD65 palmitoylation is important for the delivery of GAD65 to inhibitory synapses and suggest that impairment of GAD65 palmitoylation by mhtt may lead to altered inhibitory neurotransmission in HD.

ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

2012-02-15

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.

Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

2012-02-15

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca²⁺/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca²⁺/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

CaMKII-γ mediates phosphorylation of BAD at Ser170 to regulate cytokine-dependent survival and proliferation

2012-02-15

Phosphorylation of the BH3 (Bcl-2 homology domain 3)-only protein BAD (Bcl-2/Bcl-X_L-antagonist, causing cell death) can either directly disrupt its association with the pro-survival proteins Bcl-X_L and/or Bcl-2, or cause association of BAD with 14-3-3 proteins. In the present study, we further characterize phosphorylation of BAD at Ser¹⁷⁰, a unique site with unclear function. We provide further evidence that mutation of Ser¹⁷⁰ to a phospho-mimetic aspartic acid residue (S170D) can have a profound inhibitory effect on the pro-apoptosis function of BAD. Furthermore, mutated BAD with an alanine substitution inhibited cell proliferation, slowing progression specifically through S-phase. We identify the kinase responsible for phosphorylation at this site as CaMKII-γ (γ isoform of Ca²⁺/calmodulin-dependent kinase II), but not the other three isoforms of CaMKII, revealing an extraordinary specificity among these closely related kinases. Furthermore, cytokine treatment increased BAD-Ser¹⁷⁰-directed CaMKII-γ activity and phosphorylation of CaMKII-γ at an activating site, and CaMKII activity directed to the BAD-Ser¹⁷⁰ site was elevated during S-phase. Treating cells with a selective inhibitor of CaMKII caused apoptosis in cells expressing BAD, but not in cells expressing the BAD-S170D mutant. The present study provides support for BAD-Ser¹⁷⁰ phosphorylation playing a key role not only in regulating BAD's pro-apoptotic activity, but also in cell proliferation.

Both p110α and p110β isoforms of PI3K can modulate the impact of loss-of-function of the PTEN tumour suppressor

2012-02-15

The PI3K (phosphoinositide 3-kinase) pathway is commonly activated in cancer as a consequence of inactivation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K signalling. In line with this important role of PTEN, mice that are heterozygous for a PTEN-null allele (PTEN^+/− mice) spontaneously develop a variety of tumours in multiple organs. PTEN is a phosphatase with selectivity for PtdIns(3,4,5)P₃, which is produced by the class I isoforms of PI3K (p110α, p110β, p110γ and p110δ). Previous studies indicated that PTEN-deficient cancer cell lines mainly depend on p110β, and that p110β, but not p110α, controls mouse prostate cancer development driven by PTEN loss. In the present study, we investigated whether the ubiquitously expressed p110α can also functionally interact with PTEN in cancer. Using genetic mouse models that mimic systemic administration of p110α- or p110β-selective inhibitors, we confirm that inactivation of p110β, but not p110α, inhibits prostate cancer development in PTEN^+/− mice, but also find that p110α inactivation protects from glomerulonephritis, pheochromocytoma and thyroid cancer induced by PTEN loss. This indicates that p110α can modulate the impact of PTEN loss in disease and tumourigenesis. In primary and immortalized mouse fibroblast cell lines, both p110α and p110β controlled steady-state PtdIns(3,4,5)P₃ levels and Akt signalling induced by heterozygous PTEN loss. In contrast, no correlation was found in primary mouse tissues between PtdIns(3,4,5)P₃ levels, PI3K/PTEN genotype and cancer development. Taken together, our results from the present study show that inactivation of either p110α or p110β can counteract the impact of PTEN inactivation. The potential implications of these findings for PI3K-targeted therapy of cancer are discussed.

Effects of acutely inhibiting PI3K isoforms and mTOR on regulation of glucose metabolism in vivo

2012-02-15

In in vitro studies class-I PI3Ks (phosphoinositide 3-kinases), class-II PI3Ks and mTOR (mammalian target of rapamycin) have all been described as having roles in the regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that the class-I PI3K isoforms p110α, p110β, and to a lesser extent p110γ, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI3K activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI3K isoforms alone, or PI3K and mTOR, on glucose metabolism and food intake. In the present study impairments in glucose tolerance, insulin tolerance and increased hepatic glucose output were observed in mice treated with the pan-PI3K/mTOR inhibitors PI-103 and NVP-BEZ235. The finding that ZSTK474 has similar effects indicates that these effects are due to inhibition of PI3K rather than mTOR. The p110α-selective inhibitors PIK75 and A66 also induced these phenotypes, but inhibitors of p110β, p110δ or p110γ induced only minor effects. These drugs caused no significant effects on BMR (basal metabolic rate), O₂ consumption or water intake, but BEZ235, PI-103 and PIK75 did cause a small reduction in food consumption. Surprisingly, pan-PI3K inhibitors or p110α inhibitors caused reductions in animal movement, although the cause of this is not clear. Taken together these studies provide pharmacological evidence to support a pre-eminent role for the p110α isoform of PI3K in pathways acutely regulating glucose metabolism.

Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells

2012-02-15

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.