A molecular sensor system based on genetically engineered alkaline phosphatase.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. We show that Jaks are activated by tyrosine phosphorylation in cells that are briefly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated [or interleukin 4 (IL-4)-dependent] activation of Stat6 but can be rescued by complementation with functional Jak1. The docking sites for both Jak1 and Stat6 reside in the cytoplasmic domain of the IL-4 receptor α-chain (IL-4Rα). The glioblastoma-derived cell lines T98G, GRE, and M007, which do not express the IL-4Rα chain, fail to support Stat6 activation in response to either IL-4 or PV. Complementation of T98G cells with the IL-4Rα restores both PV-mediated and IL-4-dependent Stat6 activation. Murine L929 cells, which do not express the γ common chain of the IL-4 receptor, support PV-mediated but not IL-4-dependent Stat6 activation. Thus, Stat6 activation by PV is an IL-4Rα-mediated, Jak1-dependent event that is independent of receptor dimerization. We propose that receptor-associated constitutive PTP activity functions to down-regulate persistent, receptor-linked kinase activity. Inhibition or deletion of PTP activity results in constitutive activation of cytokine signaling pathways.

The lipid phosphatase activity of PTEN is critical for its tumor supressor function

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.

Vps41p Function in the Alkaline Phosphatase Pathway Requires Homo-oligomerization and Interaction with AP-3 through Two Distinct Domains

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein–dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

The phosphatase activity of carbonic anhydrase III is reversibly regulated by glutathiolation.

Carbonic anhydrase isozyme III (CAIII) is unique among the carbonic anhydrases because it demonstrates phosphatase activity. CAIII forms a disulfide link between glutathione and two of its five cysteine residues, a process termed S-glutathiolation. Glutathiolation of CAIII occurs in vivo and is increased during aging and under acute oxidative stress. We show that glutathiolation serves to reversibly regulate the phosphatase activity of CAIII. Glutathiolation of Cys-186 is required for phosphatase activity, while glutathiolation of Cys-181 blocks activity. Phosphotyrosine is the preferred substrate, although phosphoserine and phosphothreonine can also be cleaved. Thus, glutathiolation is a reversible covalent modification that can regulate CAIII, a phosphatase that may function in the cellular response to oxidative stress.

Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.

Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.

DNA-damaging agents induce accumulation of the tumor suppressor and G1 checkpoint protein p53, leading cells to either growth arrest in G1 or apoptosis (programmed cell death). The p53-dependent G1 arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RB). Recent studies suggest a p53-independent G1 checkpoint as well; however, little is known about its molecular mechanisms. We report that induction of a protein-serine/threonine phosphatase activity by DNA damage signals is at least one of the mechanisms responsible for p53-independent, RB-mediated G1 arrest and consequent apoptosis. When two p53-null human leukemic cell lines (HL-60 and U-937) were treated with a variety of anticancer agents, RB became hypophosphorylated, accompanied with G1 arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G1 apoptotic cells and the internucleosomal fragmentation of DNA. Addition of calyculin A or okadaic acid (specific serine/threonine phosphatase inhibitors) or zinc chloride (apoptosis inhibitor) prevented the G1 arrest- and apoptosis-specific RB dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RB dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RB and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RB dephosphorylation, which was inhibitable by calyculin A but not zinc. We propose that the RB phosphatase(s) might be one of the p53-independent G1 checkpoint regulators.

Derivation of pluripotent stem cells from cultured human primordial germ cells

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Roles of Bone Morphogenetic Protein Type I Receptors and Smad Proteins in Osteoblast and Chondroblast Differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor–like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation.

Artificial regulation of gene expression in Escherichia coli by RNase P.

Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.

Development in one dimension: the rapid differentiation of Dictyostelium discoideum in glass capillaries.

When Dictyostelium discoideum cells are drawn into a fine glass capillary, they rapidly begin the first steps toward the formation of prestalk and prespore zones. Some of the events occur within a minute or two, whereas others follow later. The cells in the front segment are actively motile and those in the hind segment are passive. The volumes of the segments are proportional for different-sized cell masses, and those proportions are the same as those found in normal slugs. When the cells are stained with the vital dye neutral red, the anterior zone becomes darker simultaneously with the formation of the division line. Green fluorescent protein expressed from a stalk-specific promoter is synthesized mostly in the anterior end. Later, this capillary prestalk zone shows a sharp increase in alkaline phosphatase activity, which is known to be characteristic of prestalk cells.

Folding and activity of circularly permuted forms of a polytopic membrane protein

The transmembrane subunit of the Glc transporter (IICBGlc), which mediates uptake and concomitant phosphorylation of glucose, spans the membrane eight times. Variants of IICBGlc with the native N and C termini joined and new N and C termini in the periplasmic and cytoplasmic surface loops were expressed in Escherichia coli. In vivo transport/in vitro phosphotransferase activities of the circularly permuted variants with the termini in the periplasmic loops 1 to 4 were 35/58, 32/37, 0/3, and 0/0% of wild type, respectively. The activities of the variants with the termini in the cytoplasmic loops 1 to 3 were 0/25, 0/4 and 24/70, respectively. Fusion of alkaline phosphatase to the periplasmic C termini stabilized membrane integration and increased uptake and/or phosphorylation activities. These results suggest that internal signal anchor and stop transfer sequences can function as N-terminal signal sequences in a circularly permuted α-helical bundle protein and that the orientation of transmembrane segments is determined by the amino acid sequence and not by the sequential appearance during translation. Of the four IICBGlc variants with new termini in periplasmic loops, only the one with the discontinuity in loop 4 is inactive. The sequences of loop 4 and of the adjacent TM7 and TM8 are conserved in all phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system transporters of the glucose family.

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.