Insulin receptor substrate-1 (IRS-1)1 is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Many of the metabolic and mitogenic effects elicited by insulin and IGF-1 are mediated and modulated by posttranslational modifications of IRS-1, and tight regulation at the posttranslational level is crucial for maintaining insulin sensitivity and controlling growth factor-induced proliferation. Following hormonal stimulation, IRS-1 is phosphorylated by the receptor tyrosine kinases creating SH2 domain docking sites for downstream binding partners including the p85 regulatory subunits of phosphatidylinositol 3-kinase, Grb2, and the tyrosine phosphatase SHP2 (PTPN11) (1). Binding of p85 phosphatidylinositol 3-kinase and Grb2 activate the PI3K/Akt and Ras-MAPK pathways, respectively, whereas binding of SHP2 results in tyrosine dephosphorylation and signal attenuation (2). Positive and negative feedback regulation by Ser/ Thr kinases, such as Akt (3), c-Jun N-terminal kinase (JNK) (4), S6K (5), and ERK (6), impact the interactions of IRS-1 with SH2 domain proteins and the receptor thereby affecting the duration and outcome of the signal. IRS-1 has been described as a central node for the integration of information regarding the nutrient and stress status of the cell (7). This information is encoded by site-specific phosphorylation by a number of kinases that regulate the specificity of effects that are elicited following receptor stimulation. Many sites of Ser/Thr phosphorylation have been identified on IRS-1, and cross-talk among Tyr and Ser/Thr phosphorylations at specific residues is evidence of dynamic and complex posttranslational regulation (8, 9). Inappropriate phosphorylation of IRS-1 resulting in the disruption of interactions of IRS-1 with binding partners From the

(3), O-GlcNAc modification is now appreciated as a nutrient-responsive mechanism for modulating signal transduction (4, 5) and transcriptional regulation (6, 7). In contrast to N-and O-linked glycosylation taking place in the endoplasmic reticulum and Golgi apparatus, reversible O-GlcNAcylation occurs in the cytoplasm and nucleus and is catalyzed by O-GlcNAc transferase (OGT), which transfers GlcNAc from UDP-GlcNAc to Ser/Thr residues, and O-GlcNAcase (OGA), which removes it. These highly conserved enzymes are expressed in all mammalian tissues (8 -11).The OGT gene encodes three splice variants differing in the number of N-terminal tetratricopeptide repeats that mediate protein-protein interactions and subcellular localization (8,12). The full-length nucleocytoplasmic form of OGT is OGlcNAc-modified, tyrosine-phosphorylated, and dynamically redistributed within the cell upon insulin stimulation (2,(13)(14)(15). Ablation of OGT is embryonically lethal or developmentally limiting in animal models (10,16,17) with the noted exception of Caenorhabditis elegans. In C. elegans, the phenotypes of oga-1 and ogt-1 null mutants suggest the enzymes are involved in macronutrient storage and life span (18 -20), and subsequent studies revealed the presence of O-GlcNAc modFrom the

No abstract

The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry combined with bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5 min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation.

Apolipoprotein H, also known as beta-2-glycoprotein I, was purified from human serum, and antiserum produced to denatured apolipoprotein H detected a cDNA clone from a lambda gt11 library derived from human liver. This cDNA coded for the complete sequence of the mature protein. The cDNA insert, along with a polymerase chain reaction product which extended the 5' end of the message, were subcloned and both strands were sequenced. The apolipoprotein H precursor was found to code for 345 amino acids, 326 of which appear in the mature protein. The deduced amino acid sequence of human apolipoprotein H differs from its rat homologue by the presence of a 48-amino acid stretch which is absent from the rat protein. The remainder of the proteins share a greater than 80% similarity. The amino acid sequence of apolipoprotein H consists largely of repeated units approximately 60 amino acids in length. These repeats are comparable to "sushi structures" found in a large number of diverse proteins, including complement components, receptors and regulators of complement activation, serum proteins, membrane-associated adhesion proteins, and other structural and catalytic proteins. Apolipoprotein H was shown to be transcribed by human hepatoma cell lines Hep 3B and Hep G2, and rat liver by detection of mRNA using northern blot analysis.