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Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127 % of normal), isoleucine (139 %), and valine (147 %) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) ␤ receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF ␤ receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF ␤ receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF ␤ receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF ␤ receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF ␤ receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domaincontaining proteins and (ii) phosphorylation of substrates other than the receptor itself.The 44-amino-acid E5 protein of bovine papillomavirus type 1 (BPV) causes morphologic and tumorigenic transformation of bovine and rodent fibroblasts in culture (7). The transformation of fibroblasts by the membrane-associated E5 protein appears to be mediated by a cellular target, the platelet-derived growth factor (PDGF) ␤ receptor. In E5-transformed mouse C127 cells, both the mature and immature precursor forms of the endogenous PDGF ␤ receptor are constitutively phosphorylated on tyrosine residues, the PDGF ␤ receptor displays increased in vitro tyrosine kinase activity, and the E5 protein is present in a stable complex with the activated PDGF ␤ receptor (34,36). Elevated levels of tyrosine phosphorylation of the endogenous PDGF ␤ receptor have been detected in E5-transformed rat and bovine fibroblasts as well (35,36).Functional studies demonstrating the importance of the PDGF ␤ receptor activation for E5 action have also been carried out. C127 cell variants that have an impaired response to PDGF respond poorly to the E5 protein (37). Furthermore, N-MuMG epithelial cells, which do not express the endogenous PDGF ␤ receptor gene, cannot be transformed by the E5 protein. However, if an exogenous PDGF ␤ receptor gene is introduced and coexpressed wit...

When expressed in PC12 cells, the platelet-derived growth factor ␤ receptor (␤PDGF-R) mediates cell differentiation. Mutational analysis of the ␤PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogenactivated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60 c-src or the persistent regulation of phospholipase C␥ was required for PC12 cell differentiation. ␤PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.The platelet-derived growth factor receptor (PDGF-R) is a transmembrane polypeptide encoding an intrinsic tyrosine kinase in its intracellular domain. Two distinct PDGF-R genes encode either an ␣ (7, 42, 47) or a ␤ (6, 19, 73) subunit. Binding of PDGF (22) induces dimerization (23) and trans phosphorylation of the ␤PDGF-R on specific tyrosine residues (35). The phosphorylated receptor initiates a series of intracellular signals which ultimately lead to cell growth (12), differentiation (20), and chemotaxis (70) depending on the cellular context.A number of tyrosines on the intracellular domain of the ␤PDGF-R are phosphorylated upon activation of the receptor and serve as recognition sites for proteins which contain Src homology 2 (SH2) domains (57). For example, the ␤PDGF-R has been shown to associate with Src family tyrosine kinases p60 c-src , p59 fyn , and p62 c-yes (39) via juxtamembrane tyrosines 579 and 581, which are in vivo phosphorylation sites (50). The SH2 domain-containing proteins p46 and p52 (Shc proteins) bind to the ␤PDGF-R at multiple phosphotyrosines including tyrosine 581 and indirectly via association with other tyrosinephosphorylated proteins (74). Phosphorylation of tyrosines 740 and 751 is critical for association of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) with the ␤PDGF-R (2, 8, 29-31). Phosphorylated tyrosine 751 also binds Nck via its SH2 binding domain (52). The GTPase-activating protein (GAP) of Ras is tyrosine phosphorylated in response to PDGF and binds to the receptor at phosphorylated 33,49). The SH2-containing phosphotyrosine phosphatase, Syp, associates with phosphorylated tyrosine 1009 (34, 41). In addition, there is evidence which suggests that the adaptor protein, Grb2, associates with the ␤PDGF-R via tyrosine 716 (1) and indirectly through Syp (44). Phosphorylated tyrosine 1009 may also influence the binding of phospholipase C␥ (PLC␥) to tyrosine 1021 (28, 59). Mutational analysis of the ␤PDGF-R has demonstrated that phosphorylated tyrosines 740 and 751, which mediate association with PI3-K, and tyrosine 1021, which mediates association with PLC␥, are necessary for the transducti...

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34 cdc2 -related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. However, substrates of CDK11 during apoptosis have not been identified. We used a yeast two-hybrid screening strategy and identified eukaryotic initiation factor 3 p47 protein (eIF3 p47) as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11 p46 ). We demonstrate that the eIF3 p47 can interact with CDK11 in vitro and in vivo, and the interaction can be strengthened by stimulation of apoptosis. EIF3 p47 contains a Mov34/JAB domain and appears to interact with CDK11 p46 through this motif. We show in vitro that the caspase-processed CDK11 p46 can phosphorylate eIF3 p47 at a specific serine residue (Ser 46 ) and that eIF3 p47 is phosphorylated in vivo during apoptosis. Purified recombinant CDK11 p46 inhibited translation of a reporter gene in vitro in a dose-dependent manner. In contrast, a kinase-defective mutant CDK11 p46M did not inhibit translation of the reporter gene. Stable expression of CDK11 p46 in vivo inhibited the synthesis of a transfected luciferase reporter protein and overall cellular protein synthesis. These data provide insight into the cellular function of CDK11 during apoptosis.

The relationship between mean platelet volume (MPV) and platelet arachidonic acid metabolism was examined by studying the ability of human platelets of different size to incorporate and metabolize tritiated arachidonic acid ([3H]AA). Platelet phospholipids were labelled with [3H]AA and the platelets were then fractionated into size-dependent subpopulations by counterflow centrifugation. The incorporation of [3H]AA increased through the fractions proportional to the MPV. After thrombin stimulation the per cent of total 3H-radioactivity released from the platelets decreased as the MPV increased. However, fractionation of the released 3H-radioactivity by HPLC (high performance liquid chromatography) demonstrated that MPV had no significant influence on the per cent of total platelet 3H-radioactivity released as cyclooxygenase products or as HETE (12-hydroxyeicosatetraenoic acid) but that the release of unmetabolized [3H]AA decreased as MPV increased. In separate experiments using unlabelled platelets the absolute release of thromboxane B2 (TXB2) after collagen- and thrombin-induced aggregation was measured by radioimmunoassay and was found to increase in proportion to the MPV. These results demonstrate that the release of arachidonic acid metabolites is qualitatively similar in platelets of different size. However, the absolute ability of platelets to incorporate arachidonic acid, convert it to active metabolites and release them is proportional to their volume. The ability of platelets to release unmetabolized arachidonic acid varies inversely with their MPV.