The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) catalytic subunit with multiple regulatory roles thought to be specified by association with different cyclin partners (Pcls). Pcl10p is one of four Pcls with little sequence similarity to cyclins involved in cell cycle control. It has been implicated in specifying the phosphorylation of glycogen synthase (Gsy2p). We report that recombinant Pho85p and Pcl10p produced in Escherichia coli reconstitute an active Gsy2p kinase in vitro. Gsy2p phosphorylation required Pcl10p, occurred at physiologically relevant sites, and resulted in inactivation of Gsy2p. The activity of the reconstituted enzyme was even greater than Pho85p-Pcl10p isolated from yeast, and we conclude that, unlike many Cdks, Pho85p does not require phosphorylation for activity. Pcl10p formed complexes with Gsy2p, as judged by (i) gel filtration of recombinant Pcl10p and Gsy2p, (ii) coimmunoprecipitation from yeast cell lysates, and (iii) enzyme kinetic behavior consistent with Pcl10p binding the substrate. Synthetic peptides modeled on the sequences of known Pho85p sites were poor substrates with high K m values, and we propose that Pcl10p-Gsy2p interaction is important for substrate selection. Gel filtration of yeast cell lysates demonstrated that most Pho85p was present as a monomer, although a portion coeluted in high-molecular-weight fractions with Pcl10p and Gsy2p. Overexpression of Pcl10p sequestered most of the Pho85p into association with Pcl10p. We suggest a model for Pho85p function in the cell whereby cyclins like Pcl10p recruit Pho85p from a pool of monomers, both activating the kinase and targeting it to substrate.

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Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey MichaelisMenten kinetics for hydrolysis of retinyl palmitate with K m values of about 1 lM and specific activities between 3 and 8 nmolAEmin )1 AEmg )1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.Keywords: retinyl palmitate hydrolase, carboxylesterase, mass spectrometry, rat, retinol, vitamin A.Vitamin A metabolism [1,2] is a significant area of research because of its diverse role in the regulation of gene expression through retinoic acid receptors. Dietary intake of vitamin A from animal food products is mainly in the form of retinyl esters and retinol, and from plant food products such as provitamin A or b-carotenes. Retinyl esters are converted to retinol in the intestine. After dietary uptake, retinol is converted to retinyl esters in intestinal mucosa and packaged into chylomicrons. These are partially processed during circulation to chylomicron remnants, which contain retinyl esters. Chylomicron remnants are rapidly cleared from circulation by liver hepatocytes where the retinyl esters are hydrolyzed by retinyl ester hydrolases to retinol. The retinol product can either undergo oxidation to retinoic acid for signaling or be secreted into circulation as a complex with retinol binding protein. After meeting the tissue needs, excess retinol is stored in hepatic stellate cells by conversion to retinyl esters (mostly as retinyl palmitate). The stored retinyl esters are the primary vitamin A reservoir in the body and can be mobilized by hydrolysis to retinol by retinyl ester hydrolases. Hence, retinyl ester hydrolases play very important roles in a variety of cells and tissues to...

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