Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. AUl of the amino acid substitutions were clustered in the amino-terminal domain ofthe protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.Protein phosphorylation is a major regulatory mechanism for the control of cellular metabolism, growth, and differentiation (1-3). A number of protein kinases have been identified that specifically phosphorylate various protein substrates. Phosphoserine and phosphothreonine protein phosphatases have been broadly categorized as either type 1 or type 2 based on their sensitivity to inhibitors and substrate specificity (4). The type 2 phosphatases have been further divided into three groups termed 2A, 2B, and 2C (5). Little structural information has been available for the catalytic subunits of the protein phosphatases, but protein sequencing (6) and isolation of appropriate cDNAs (7-9) have indicated there are significant homologies between the type 1 and type 2 protein phosphatases, suggesting they have been highly conserved in primary sequence.By using a partial cDNA probe to bovine phosphatase 2A (7) Tris HCI (pH 8.0), 0.9 M NaCi, 6 mM EDTA, 5 x Denhardt's solution (0.1% Ficoll/0.1% polyvinylpyrrolidone/0.1% bovine serum albumin), 10% (wt/vol) dextran sulfate, 0.1% NaDodSO4, and the radiolabeled probe (6 x i0' cpm/ml).The nitrocellulose filters were washed twice in 2 x SSC at room temperature for 10 min and once at 530C for 30 min.(1 x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0.)Recombinant phage giving positive signals were plaquepurified. DNA prepared from positive recombinant phage was isolated and the inserts were subcloned into pUC18 and M13mpl8 vectors. At this point, restriction analysis of the clones was carried out by incubation with the appropriate restriction endonucleases followed by electrophoretic analysis in agarose gels (12).The nucleotide sequence of the clones was established after subcloning the appropriate restriction fragments into M13mpl8 or M13mpl9 phage (13). All sequencing reactions were performed by the dideoxy chain-termination method (14) with modified T7 DNA polymerase (15), and both strands were sequenced in all cases. The sequencing of the HL-1 and HL-14 clones was also performed with the Cyclone system (International Biotechnologies, New Haven, CT) to progressively del...

Abstract. The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUTI) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera eDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HAtagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-'/and CHO cells.

The protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following exper-imental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophos- The insulin receptor is one of a number of growth factor receptors with intrinsic tyrosine kinase activity that can be activated upon binding of appropriate peptide ligands (1). Binding of insulin to its receptor also causes rapid phosphorylation of the tyrosine residues of the insulin receptor (3 subunit itself (2-4), which in turn further activates the tyrosine kinase activity (5, 6). In intact cells, insulin causes receptor phosphorylation on tyrosine as well as serine and threonine residues (2). Protein kinase C or protein kinases activated by this enzyme can phosphorylate the serine and threonine residues of the insulin receptor in intact cells (7,8). Stimulation of serine phosphorylation of the insulin receptor by phorbol esters appears to correlate with inhibition of the insulin-stimulated tyrosine kinase activity ofthe receptor (8).Thus, phosphorylation ofthe serine and threonine residues of the insulin receptor may regulate insulin signaling by modulating receptor tyrosine kinase activity. Several laboratory groups have reported the detection of an insulin-stimulated and receptor-associated serine/ threonine kinase activity using the receptor itself as substrate, in partially purified (9-14) or affinity-purified (15) insulin receptor preparations.

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3(+)(/)(+)) and Tff3-deficient (Tff3(-)(/)(-)) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3(-/-) mice is significantly prolonged in comparison to Tff3(+/+) mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3(+/+) and Tff3(-/-) mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

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