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  • br Materials and methods br Results br Discussion Adiponecti

    2019-09-07


    Materials and methods
    Results
    Discussion Adiponectin is one of the most studied adipocytokines. Its release from adipocytes is down-regulated in obesity and other adverse metabolic conditions leading to decreased serum concentrations (Turer and Scherer, 2012). Several factors and conditions are involved in the inhibition of adiponectin expression such as other inflammatory factors, ER stress (Liu and Liu, 2014), and mitochondrial dysfunction (Liu and Liu, 2014). Since stress-related kinases have been shown to modulate adipocyte specific processes, like secretion and stability of adipocytokines (Kondo et al, 2011, Li et al, 2013, Liu, Liu, 2010, Park et al, 2004, Tang et al, 2005) we decided to study the interplay of CK1 isoforms and adiponectin. Phosphorylation mediated by cellular kinases is considered to be one of the most common and reversible covalent posttranslational modifications (Fischer, 2013). It was described in literature that adiponectin is subject to posttranslational modifications (Liu, Liu, 2014, Simpson, Whitehead, 2010, Wang et al, 2008). Four conserved lysine residues residing within the collagen-like domain (Lys65, Lys68, Lys77, and Lys101) can be hydroxylated and subsequently glygosylated and both modifications are necessary for the formation of HMW complexes out of adiponectin trimers (Liu, Liu, 2014, Richards et al, 2006, Wang et al, 2002, Wang et al, 2008). Hydroxylation of several proline residues residing in the collagen-like domain has also been reported (Richards et al., 2006). Furthermore, complex formation of adiponectin is mainly due to the formation of an intermolecular disulfide bond (for overview see Liu and Liu, 2014). Amino transferase residues within the N-terminus of adiponectin are required for this covalent bond, which stabilizes the three-dimensional structure of the protein. Our data demonstrate for the first time that adiponectin is a phosphoprotein and that site-specific phosphorylation is involved in the regulation of complex formation. We provide several lines of evidence supporting this statement: first, a computer-aided search for putative phosphorylation sites indicated that several amino acids within the adiponectin protein are potential targets for intracellular kinases (among them PKC, PKA, Akt, and CK1). Second, kinase assays revealed that recombinant adiponectin is highly phosphorylated by CK1δ in vitro. Third, measuring phosphate incorporation into GST–adiponectin mediated by CK1δ revealed that at least 2 moles of phosphate were incorporated per mol substrate. This clearly indicates that there exists more than one phosphorylation site for CK1δ within full length adiponectin. According to the results of the computer-aided search and our first in vitro data, main phosphorylation sites for several cellular kinases lie within aa 168–244 in the C-terminal domain of adiponectin. The incorporation of 2–3 moles of phosphate into the GST–adiponectin168–244 fusion protein points to the existence of at least two phosphorylation sites being targeted by CK1δ. Accordingly, two dimensional phosphopeptide mapping confirmed the existence of two major phosphorylation sites within aa 168–244. Phosphoamino acid analysis confirmed serine as well as threonine residues to be targets of phosphorylation by CK1δ. Ablation of putative transferase serine/threonine phosphorylation sites within aa 168–244 by substitution with the neutral aa alanine showed that serine 174 and threonine 235 are targeted by CK1δ. Multimer formation is an important mechanism to regulate the biological activity of proteins. For example, multimerization of surfactant protein-D (SP-D) enhances viral aggregation, precipitation and neutrophil uptake of Influenza A virus (Hartshorn et al, 1996, Hartshorn et al, 1997, Hartshorn et al, 1998). Furthermore, binding of the multifunctional glycoprotein vitronectin (VN) to collagen is enhanced by increased multimerization of VN (Sano et al., 2007). The binding efficacy to maltose or liposomes differs among different multimer species of adiponectin, and its multimer distribution shifts to smaller multimers in bronchoalveolar lavage fluid from patients with pulmonary alveolar proteinosis and pollen allergies (Hickling et al, 1998, McCormack et al, 1997, Wang et al, 2002, Wang et al, 2004, Wang, Burke, 2008). As another example, von Willebrand factor forms huge multimers connected via disulfide bonds. Its biological activity depends on multimer size (Xie et al., 2000) and is mediated by phosphorylation (Bodnar et al., 2002).