Our data demonstrate for the

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 Cy7 carboxylic acid (non-sulfonated) analysis confirmed serine as well as threonine residues to be targets of phosphorylation by CK1δ. Ablation of putative 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). Several studies suggest that the beneficial metabolic effects of adiponectin in humans are primarily mediated by its HMW isoform. Increases in the ratio of HMW to total adiponectin, but not the total adiponectin level, correlated well with improved insulin sensitivity during treatment with the insulin sensitizing drug thiazolidinediones in both diabetic mice and patients with T2D (Pajvani et al, 2004, Waki et al, 2003). Likewise, prospective studies suggested that serum HMW adiponectin is a better marker than total adiponectin in the prediction of insulin resistance and the metabolic syndrome (Hara et al., 2006), T2D (Nakashima et al, 2006, Retnakaran et al, 2007), and endothelial dysfunction (Torigoe et al., 2007). In line with these epidemiological data, genetic evidence supports the role of HMW adiponectin as the major insulin-sensitizing form in humans. Two rare genetic mutations (G84R and G90S) within the collagenous domain led to extremely low levels of HMW adiponectin and were closely associated with insulin resistance and T2D (Waki et al., 2003). Likewise, two mutations in the globular domain (R112C and I164T) lead to a failure in the assembly of trimer and were associated with hypoadiponectinemia (Waki et al., 2003). Whether point mutations lead to conformational changes causing an aberrant phosphorylation of adiponectin impairing HWM formation needs to be further elucidated (Bueno et al., 2014).