This auxin insensitivity can also be observed when wild-type seedlings are treated with the NAE inhibitor MLN4924, which blocks NEDD8 conjugation in an MLN4924 concentration-dependent manner (Brownell et al., 2010; Hakenjos et al., 2011). RESISTANT1 (AXR1), a subunit of the heterodimeric CP671305 NEDD8 E1 activating enzyme, as a NEDD8-modified protein in mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins. INTRODUCTION NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8), in also known as RUB (RELATED TO UBIQUITIN), is an evolutionarily conserved 8-kD protein closely related to ubiquitin (Rao-Naik et al., 1998; Hochstrasser, 2009). Like ubiquitin, NEDD8 is usually conjugated to substrate proteins through an enzymatic cascade that includes the E1 NEDD8 activating enzyme (NAE); in Arabidopsis, NAE is usually a heterodimer of AXR1 (AUXIN RESISTANT1) or AXL (AXR1-LIKE) and ECR1 (E1 C-TERMINAL RELATED1). The NEDD8-conjugating cascade also includes an E2 conjugating enzyme; in Arabidopsis, this is RUB1 CONJUGATING ENZYME1 (RCE1; Pozo et al., 1998; del Pozo and Estelle, 1999; del Pozo et al., 2002; Dharmasiri et al., 2007; Woodward et al., 2007). NEDD8 is usually ultimately conjugated to its protein substrate with the help of E3 NEDD8 ligases like RBX1 (RING BOX1), a constitutive subunit of cullin-RING E3 ubiquitin ligases (CRLs), and DEFECTIVE IN CULLIN NEDDYLATION (DCN; Gray et al., 2002; Duda et al., 2008; Kurz et al., 2008). The CP671305 cullin subunits of CRLs are the best-characterized substrates for NEDD8 conjugation (neddylation) (Duda et al., 2008; Huang et al., 2008). Cullin neddylation is usually promoted by the CRL core subunit RBX1 and required for the assembly of functional CRL complexes that ubiquitylate their cognate substrate proteins to target them for degradation by the 26S proteasome (Gray et al., 2002; Duda et al., 2008). CRL function and protein complex assembly are antagonized by cullin deneddylation through the COP9 signalosome (CSN) (Schwechheimer et al., 2001; Wei et al., 2008; Schwechheimer and Isono, 2010; Lingaraju et al., 2014). Arabidopsis mutants for all those eight CSN subunits have been described, including mutants for the paralogous proteins CSN5A and CSN5B, which are the deneddylating subunits of CSN (Gusmaroli et al., 2004, 2007; Dohmann et al., 2005). Whereas loss-of-function mutants display the strong characteristic constitutively photomorphogenic (cop) phenotype and accumulate cullins in their NEDD8-modified form, mutants partially impaired in CSN function, such as and genes (Bostick et al., 2004) or mutants defective in both paralogous subunits of the NAE, and single mutants, undergo largely normal embryo differentiation but have substantial growth defects, including a strong insensitivity to the phytohormone auxin when grown on medium made up of auxin concentrations that inhibit root growth in the wild type (Lincoln et al., 1990; Leyser et al., 1993; Schwechheimer et al., 2002). The auxin insensitivity of the mutants can be explained by impaired functionality of their cognate E3 ligase SCFTIR1 and related CRLs and, consequently, an inability to degrade the auxin-labile AUX/IAA repressor proteins such as AXR2 and AXR3 (Gray et al., 2001). This auxin insensitivity can also CP671305 be observed when wild-type seedlings are treated with the NAE inhibitor MLN4924, which blocks NEDD8 conjugation in an MLN4924 concentration-dependent manner (Brownell et al., 2010; Hakenjos et al., 2011). Auxin-insensitive root growth is usually thus an indicator for defects in neddylation and SCFTIR1 function. Importantly, weak mutants of CSN such as and mutants also display this phenotype, suggesting that an adequate balance of neddylation and deneddylation CDH5 is required for proper CRL and SCFTIR1 function (Schwechheimer et al., 2001; Gusmaroli et al., 2004, 2007; Dohmann et al., 2005). Ubiquitin and ubiquitin-like modifiers such as SMALL UBIQUITIN-LIKE MODIFIER (SUMO) change hundreds of distinct target proteins and thereby affect protein activity or fate (Miller and Vierstra, 2011; Vierstra, 2012; CP671305 Kim et al., 2013). Therefore, it is surprising that, to date, only cullins have been recognized as bona fide NEDD8 modification substrates. For NEDD8, a number of non-cullin NEDD8-modified proteins have previously been identified, mainly in animal systems (Xirodimas, 2008; Mergner and Schwechheimer, 2014; Enchev et al., 2015), but recent observations of nonspecific crosstalk between the.
- Next Furthermore, we suspect at least two mechanisms involving TGZ regulation of pY397FAK, since at 10 M, TGZ treatment inhibited pY397FAK however, not PTEN translocation
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- Abbreviations: CON, control; CANA, canagliflozin; AMPK, AMP-activated proteins kinase; ACC, acetyl-CoA carboxylase
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