Sprouty2-mediated inhibition of fibroblast growth factor signaling is modulated by the protein kinase DYRK1A.

Raf-MEK-extracellular signal-regulated kinase (Erk) signaling initiated by growth factor-engaged receptor tyrosine kinases (RTKs) is modulated by an intricate network of positive and negative feedback loops which determine the specificity and spatiotemporal characteristics of the intracellular signal. Well-known antagonists of RTK signaling are the Sprouty proteins. The activity of Sprouty proteins is modulated by phosphorylation. However, little is known about the kinases responsible for these posttranslational modifications. We identify DYRK1A as one of the protein kinases of Sprouty2. We show that DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, we identify Thr75 on Sprouty2 as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhanced the repressive function of Sprouty2 on fibroblast growth factor (FGF)-induced Erk signaling. Further supporting the idea of a functional interaction, DYRK1A and Sprouty2 are present in protein complexes in mouse brain, where their expression overlaps in several structures. Moreover, both proteins copurify with the synaptic plasma membrane fraction of a crude synaptosomal preparation and colocalize in growth cones, pointing to a role in nerve terminals. Our results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2.

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets.

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.

Intracellular trafficking during liver regeneration. Alterations in late endocytic and transcytotic pathways.

BACKGROUND/AIMS: Liver growth, induced by partial hepatectomy of the organ is a precisely regulated process during which a radical reorganisation of metabolism occurs as the hepatocytes become committed to enter the cell cycle. Recent studies have shown the importance of the endocytic compartment in the control of lipid and protein intracellular trafficking but also in the control of the signal transduction events, which eventually will trigger the initiation of DNA synthesis and the subsequent cell division. METHODS: We isolated endosomes at different times after partial hepatectomy in male rats and compared with endosomes isolated from sham-operated animals. Also, bile was collected and analysed by 2D-gel electrophoresis. RESULTS: The amount of late endosomes isolated from regenerating livers decreased, concomitant with decreased cathepsin D specific enzyme activity. Furthermore, secretion of horseradish peroxidase, pIgA and transferrin increased in the pre-replicative phase of liver regeneration. CONCLUSIONS: At the early stages of liver regeneration, the hepatocellular transport pathway towards degradation (late endosomes and lysosomal pathway) decreases, but the transcytosis and the bile secretion of several major proteins increases.