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D J Takemoto

Publications and source records attributed to D J Takemoto.

At least 19 recordsLinked to original sources
PubMed2001

PKC-gamma phosphorylation of connexin 46 in the lens cortex.

PURPOSE: To identify the role of PKC-gamma in control of and phosphorylation of connexin 46 (Cx46) in the lens cortex. METHODS: The association between PKC-gamma and Cx46 was determined by co-immunoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-gamma were determined using Western blots, PKC activity assays, and inhibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors. RESULTS: Co-localization of PKC-gamma and Cx46 was observed in the bow regions and cortical regions of rat lens. PKC-gamma was not observed in the nuclear region and Cx46 was not observed in the epithelial layer. PKC-alpha was not found in lens cortex or nuclear regions. PKC-gamma could be co-immunoprecipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-gamma specific pseudosubstrate inhibitor caused a 73% inhibition of serine phosphorylation on Cx46 at 1 microM, and, 36% inhibition of threonine phosphorylation at the same concentration. Inhibition of phosphorylation of Cx46 with PKC-alpha pseudosubstrate inhibitor was not observed. CONCLUSIONS: PKC-gamma may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-gamma in control of lens cortical gap junctions is suggested.

Animals↗
PubMed2001

PKCalpha and PKCgamma overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line.

PURPOSE: The overexpression of PKCalpha or PKCgamma for extended periods of time causes the formation of lentoid bodies in the N/N 1003A rabbit lens epithelial cell line. To determine how differentiated the lentoid bodies are, we have looked for alphaA-, alphaB-, beta-, and gamma-crystallin levels in lentoid bodies after 4 and 8 weeks of lentoid body development. METHODS: Cells overexpressing PKCalpha or PKCgamma were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid bodies were fixed and stained with PKCalpha or PKCgamma antibodies along with either alphaA-, alphaB-, beta-, or gamma-crystallin antisera and viewed under a confocal microscope. Lentoid bodies were harvested in lysis buffer and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was probed with either alphaA-, alphaB-, beta-, or gamma-crystallin antibodies for 12 h. Total RNA from lentoid bodies was isolated and 5 microg of total RNA was transcribed to first-strand cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis. RESULTS: alphaB-crystallin was present in normal N/N 1003A cells and the lentoid bodies formed from PKCalpha and PKCgamma overexpression. alphaA-crystallin was only detectable in lentoid bodies after PKCalpha or PKCgamma overexpression. RT-PCR was able to detect beta-crystallin expression while the Western blot analysis and immunocytostaining detected small amounts of beta-crystallin protein. No gamma-crystallin expression was noted in these lentoid bodies. CONCLUSIONS: Overexpression of PKCalpha or PKCgamma in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not only alphaB-crystallin but alphaA- and beta-crystallin. These results suggest a role for PKCs in lens epithelial cell differentiation to a fiber cell.

Animals↗
PubMed2001

Protein kinase C alpha and gamma in N/N 1003A rabbit lens epithelial cell differentiation.

PURPOSE: To determine if Protein Kinase C (PKC) plays a role in the initiation of lens epithelial cell differentiation into a lens fiber cell. METHODS: PKCalpha or PKCgamma was overexpressed in N/N 1003A lens epithelial cells for up to 7 days. Phase contrast microscopy was used to observe morphological changes associated with PKCalpha or PKCgamma overexpression. Cell cycle changes in cells overexpressing PKCalpha or PKCgamma were measured using acridine orange staining and flow cytometry. Crystallin levels in cells overexpressing PKCalpha or PKCgamma were measured using Western blots and RT-PCR. RESULTS: Significant differences in cell cycling were observed between untransfected cells and those overexpressing PKCalpha or PKCgamma. Overexpression of PKCalpha and PKCgamma caused the cells to lose their epithelial-like appearance and elongate. alphaB-crystallin expression was detected in all the samples while alphaA-crystallin was detected only in cells after 7 days of PKCalpha or PKCgamma overexpression. CONCLUSIONS: The observations that alphaA-crystallin is only found in N/N 1003A cells overexpressing PKCalpha or PKCgamma for 7 days along with the finding that a block in the G0/G1 phase of the cell cycle and the consequent morphological changes are observed, indicate that PKCalpha and PKCgamma may have a role in the initiation of differentiation in lens epithelial cells.

Acridine Orange↗
PubMed1998

Decreases in Raf-1 levels in galactosaemic lens epithelial cells are partially reversed by myo-inositol.

Changes in the amounts and localization of protein kinase C (PKC) isoforms occur in galactosaemic lens epithelial cells. A link between PKC changes and myo-inositol depletion has been suggested. Raf-1, a component of a Ras pathway, is a substrate for PKC. Raf-1 levels were measured in galactosaemic lens epithelial cells grown with or without myo-inositol. Raf-1 levels were measured by densitometric scanning of Western blots from cells grown with or without 40 mmol/l galactose or 40 mmol/l galactose plus 1.0 micromol/l myo-inositol for 1, 3, 5 or 7 days. Scans were compared to those for PKCalpha, an isoform of PKC and to 14-3-3, a protein which binds to Raf-1. Cell growth was quantitated by thymidine incorporation. Raf-1 levels were decreased in bovine lens epithelial cells after 3, 5 or 7 days (33% of control) of growth in 40 mmol/l galactose. Addition of 1 micromol/l myo-inositol reversed this decrease at day 3, but not after 5 or 7 days of growth in 40 mmol/l galactose. PKCalpha and 14-3-3 levels were not affected by galactose. The decrease in Raf-1 was not a result of cell growth as measured by thymidine incorporation. These results suggest that Raf-1 levels are decreased during galactosaemia. This was only partially reversed by the addition of myoinositol.

Animals↗
PubMed1996

Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit.

The inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE alpha beta gamma 2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDE alpha beta catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDE gamma by direct interaction with PDE gamma. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J: Biol. Chem. 269, 9850-9856] has shown that PDE gamma is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDE gamma to inhibit PDE alpha beta catalytic activity. Here we report that transducin is less effective in activation of PDE alpha beta (gamma p)2 (a complex of PDE alpha beta with phosphorylated PDE gamma, PDE gamma p) than PDE alpha beta gamma 2. PDE gamma p also increases the rate constant of GTP hydrolysis of transducin (from 0.16 S-1 for non-phosphorylated PDE gamma to 0.21 s-1 for PDE gamma p). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Acridine orange differential staining of total DNA and RNA in normal and galactosemic lens epithelial cells in culture using flow cytometry.

The lens epithelial cells are a primary site of involvement in galactosemia. Changes in their size, shape and proliferative capacity have been observed upon exposure to high galactose. In this report, changes in the cell cycle pattern of normal and galactosemic lens epithelial cells were examined by use of flow cytometry. Both changes in DNA and RNA were observed using the fluorochrome, acridine orange. Under the appropriate conditions acridine orange can be used to differentiate double-stranded DNA from single-stranded RNA. Using this approach, the DNA and RNA of normal and galactosemic (1, 4, or 7 days) lens epithelial cells can be compared. The results indicate that lens epithelial cells, when exposed to 40 mM galactose media or 30 mM glucose for 7 days, are induced to enter mitosis. Mannitol did not mimic these results. Changes in the cell cycle pattern were not observed when the cells were treated for 1 or 4 days. Although higher numbers of cells in mitosis were observed after 7 days exposure to 40 mM galactose, a correlation between proliferation, as measured by 3H-thymidine uptake, and mitosis was not possible. Apoptosis was evaluated as a possible explanation for these results. The changes in the DNA staining pattern could be use to monitor lens epithelial cells during galactosemia.

Acridine Orange↗
PubMed1994

Functional effect of phosphorylation of the photoreceptor phosphodiesterase inhibitory subunit by protein kinase C.

In rod outer segments the light activation of cGMP phosphodiesterase (PDE alpha beta gamma 2) is accomplished by removal of the gamma inhibitory subunit (PDE gamma) from the PDE alpha beta catalytic subunits. A light activation of the inositol signaling pathway also occurs, but there is little information linking these two signal transduction pathways. Here we report that protein kinase C (PKC) purified from bovine rod outer segment phosphorylates the bovine PDE gamma with incorporation of 0.9 +/- 0.1 mol of phosphate/mol of PDE gamma. Phosphorylation of PDE gamma increases its ability to inhibit PDE alpha beta catalytic activity (trypsin-activated PDE, tPDE) with an IC50 for phosphorylated PDE gamma of 26 +/- 4 pM and an IC50 of 60 +/- 5 pM for unphosphorylated PDE gamma. Inhibition of tPDE by PDE gamma is characterized by two values of Kd, Kd1 = 34 pM and Kd2 = 760 pM. Phosphorylation of PDE gamma by PKC eliminates the functional heterogeneity of the PDE gamma population resulting in a single value of Kd = 23 pM. Free PDE gamma (without PDE alpha beta catalytic subunits) is a better substrate for PKC than PDE gamma in a complex with PDE alpha beta. Phosphorylation of free PDE gamma by PKC is characterized by a value of Vmax = 1,550 +/- 148 units/mg (Km = 21.0 +/- 1.9 microM). In contrast, phosphorylation of PDE gamma in PDE alpha beta gamma 2 complex has two values of Vmax, Vmax1 = 0.3 +/- 0.1 units/mg of PDE gamma (Km1 = 0.4 +/- 0.2 microM) and Vmax2 = 0.7 +/- 0.2 units/mg of PDE gamma (Km2 = 4.6 +/- 0.9 microM). ROS PKC phosphorylates Thr35 in PDE gamma. We have previously reported (Morrison, D. F., Rider, M. A., and Takemoto, D. J. (1987) FEBS Lett. 222, 266-270; Lipkin, V. M., Udovichenko, I. P., Bodarenko, V. A., Yurovskaya, A. A., Telnykh, E. V., and Skiba, N. P. (1990) Biomed. Sci. (Lond.) 1, 305-308) that the central fragment of PDE gamma (24-45) is responsible for binding to PDE catalytic subunits. The new data suggests that this region of PDE gamma also includes the site for phosphorylation by PKC and that phosphorylation increases the ability of PDE gamma to inhibit PDE catalytic activity. This altered regulation of visual transduction may play a role in desensitization or light adaptation.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

Identification of a binding site on retinal transducin alpha for the phosphodiesterase inhibitory gamma subunit.

Transducin alpha (T alpha) activates retinal rod cyclic GMP phosphodiesterase (PDE) by interacting with and removing the inhibitory PDE gamma subunit. A T alpha-PDE gamma complex can be isolated in vitro, and our previous work [Morrison, Rider and Takemoto (1987) FEBS Lett. 222, 266-270; Morrison, Cunnick, Oppert and Takemoto (1989) J. Biol. Chem. 264, 11671-11681] has identified a region of PDE gamma, residues 24-45, that binds to T alpha. The C-terminal region of PDE gamma is the site that interacts with PDE alpha/beta and inhibits catalytic function. The site on T alpha that binds to the PDE gamma 24-45 region has not been identified. Synthetic peptides (15-mers) which span the bovine T alpha sequence were tested for binding to purified recombinant PDE gamma using a solid-phase assay. The peptides were also tested for ability to activate a PDE complex. We have identified a region, residues 250-275 of T alpha, which shows a high affinity of PDE gamma and for the PDE gamma (24-45) binding peptide. The peptide did not bind to the C-terminal residues 50-87 of PDE gamma. Likewise, a region of T alpha, 1-25 did not exhibit high-affinity binding to PDE gamma or to the 24-45 PDE gamma peptide. Specific binding of the 250-275 peptide to PDE gamma was confirmed by its ability to compete with T alpha binding to PDE gamma, although a higher concentration was required (10x). The T alpha-(250-275) peptide activated a fully inhibited PDE alpha beta gamma 2 complex in a dose-dependent manner. These results suggest that a region on T alpha that recognizes the PDE gamma-binding site is found within residues 250-275 of T alpha.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase.

The cyclic GMP phosphodiesterase (PDE) of retinal rods plays a key role in phototransduction and consists of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Here we report that PDE alpha and PDE gamma are phosphorylated by protein kinase(s) C (PKC) from brain and rod outer segments (ROS). These same two types of PKC also phosphorylate PDE alpha in trypsin-activated PDE (without PDE gamma). In contrast, cyclic-AMP-dependent protein kinase catalytic subunit phosphorylates both PDE alpha and PDE beta, but not PDE gamma. This kinase does not phosphorylate trypsin-activated PDE. The synthetic peptides AKVISNLLGPREAAV (PDE alpha 30-44) and KQRQTRQFKSKPPKK (PDE gamma 31-45) inhibited phosphorylation of PDE by PKC from ROS. These data suggest that sites (at least one for each subunit) for phosphorylation of PDE by PKC are localized in these corresponding regions of PDE alpha and PDE gamma. Isoenzyme-specific PKC antibodies against peptides unique to the alpha, beta, gamma, delta, epsilon and zeta isoforms of protein kinase C were used to show that a major form of PKC in ROS is PKC alpha. However, other minor forms were also present.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Protein kinase C in galactosemic and tolrestat-treated lens epithelial cells.

Using isozyme-specific anti-peptide antisera against peptides from the alpha-, beta-, gamma-, delta-, epsilon-, and zeta-isoforms of brain protein kinase C (PKC), we have identified proteins in bovine lens epithelial cells, in culture, that were reactive with these antisera. Western blots of lens epithelial cell homogenates showed that PKC-alpha antisera reacted with a major protein, and PKC-gamma antisera reacted with a minor protein. When the lens epithelial cells were cultured in media supplemented with 40 mM galactose, to model the conditions of sugar cataracts, a decrease in PKC-gamma, but not in PKC-alpha was observed. These were normalized if the cells were cultured in 40 mM galactose media supplemented with an inhibitor of aldose reductase, Tolrestat (10 microM). These results suggest that changes in PKC isoforms occur in the galactosemic diabetic state.

Aldehyde Reductase↗
PubMed1992

Carriers of the mouse rd gene have reduced levels of the beta subunit of the retinal cyclic GMP phosphodiesterase.

Polyclonal antipeptide antisera have been utilized to quantitate the amount of retinal rod outer segment cGMP phosphodiesterase alpha and beta catalytic subunits present in retinas from C57BL/6J mice which are normal or carriers for the rd gene defect. Results suggest that the quantity of PDE-beta subunit is reduced in carrier mice while PDE-alpha and PDE-gamma are not affected. In 21-day-old mice, the PDE-beta was reduced by about one-half while adult carrier mice had even more reduced levels of PDE-beta. Since PDE alpha was not reduced, this suggests that synthesis of PDE alpha and PDE beta may not be coordinately controlled.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Domain mapping of the retinal cyclic GMP phosphodiesterase gamma-subunit. Function of the domains encoded by the three exons of the gamma-subunit gene.

Retinal rod-outer-segment phosphodiesterase (PDE) is a heterotetramer consisting of two similar, but not identical, catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). Previously, we have reported that the site of PDE alpha/beta interaction with PDE gamma is located within residues 54-87 [Cunnick, Hurt, Oppert, Sakamoto & Takemoto (1990) Biochem. J. 271, 721-727]. The site for PDE gamma interaction with transducin alpha (T alpha) was found to encompass residues 24-45 of PDE gamma [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. In order to identify binding sites and other functional domains of PDE gamma, the three peptides which are encoded by the three exons of the PDE gamma gene were synthesized chemically. These exons encode for residues 1-49, 50-62 and 63-87 of bovine PDE gamma [Piriev, Purishko, Khramtsov & Lipkin (1990) Dokl. Akad. Nauk. SSSR 315, 229-230]. The peptide encompassing residues 63-87 was inhibitory in a PDE assay, whereas peptides 1-49 and 50-62 had no effect. However, both peptides 1-49 and 63-87 bound to PDE alpha/beta in a solid-phase binding assay. Only peptide 1-49 bound to T alpha.GTP[S] (GTP[S] is guanosine 5'-[gamma-thio]triphosphate). These data confirm that the inhibitory region of PDE gamma is encoded by exon 3 (residues 63-87), whereas a separate binding site for PDE alpha/beta and for T alpha.GTP[S] is encoded by exon 1 (residues 1-49). To study further the structure-function relationship of PDE gamma, this entire protein and two mutants were chemically synthesized. One mutant (-CT) lacked residues 78-87, whereas another replaced tyrosine-84 with glycine (TYR-84). Whereas the synthetic PDE gamma inhibited PDE alpha/beta catalytic activity, the -CT and TVR-84 mutants did not. All three synthetic proteins bound to both PDE alpha/beta and and T alpha.GTP[S]. These data confirm the presence of an alternative binding site on PDE gamma and demonstrate the importance of tyrosine-84 in PDE gamma inhibitory activity.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Identification of the retinal cyclic GMP phosphodiesterase inhibitory gamma-subunit interaction sites on the catalytic alpha-subunit.

Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDE gamma was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDE alpha used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDE gamma; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radio-immunoassays and PDE alpha peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90, respectively) were able to bind PDE gamma. Additionally, peptides corresponding to the PDE alpha region 453-534 bound PDE gamma in the binding assay. This suggests that several regions on PDE alpha interact with the PDE gamma inhibitor. While some regions may be involved in binding to PDE gamma, other sites may be involved in PDE gamma inhibition of catalytic activity. Our results suggest that the major regions of PDE alpha that interact with PDE gamma reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Sequence analysis of three retinal phosphodiesterases (rod outer segment alpha, beta, and cone outer segment alpha') revealed the highest region of dissimilarity in the N and C termini.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Antibody indications of secondary and superimposed retinal hypersensitivity in retinitis pigmentosa.

Antibody reactions with recognized retinopathy-inducing retinal antigens may be interpreted to reflect ongoing autoimmune events responsible for some forms of vision loss. We sought evidence of secondary and superimposed retinal hypersensitivity indicated by such antibody reactivity in a random group of patients with retinitis pigmentosa. We identified patterns of immunologic reactivity within members of a group of 52 patients with retinitis pigmentosa, which suggests some patients with retinitis pigmentosa may experience consequential superimposed retinal hypersensitivity. Identifying subgroups of patients with retinitis pigmentosa who exhibit indications of retinal hypersensitivity to known uveitopathogenic retinal proteins may permit the reduction of their rate of retinal degradation by immunomodulation.

Adult↗
PubMed1991

Identification of the gamma-subunit interaction sites in the retinal cyclic-GMP phosphodiesterase beta-subunit.

Using synthetic peptides, the identification of the retinal cyclic-GMP phosphodiesterase (cGMP PDE) interaction sites for the inhibitory gamma-subunit in the catalytic alpha-subunit were recently localized to residues #16-30 and 78-90 in the alpha-subunit (1). In this study, a binding radioimmunoassay (RIA) showed a weak interaction between PDE gamma and PDE beta subunits in PDE beta residues #15-34, and stronger interaction sites were found in residues #91-110 and 211-230. Sequence comparison between PDE alpha and PDE beta illustrate some differences in these regions, particularly in PDE alpha 16-30 and PDE beta 15-34 regions. Differences in interaction sites in PDE alpha and PDE beta for PDE gamma may account for the differences in affinities observed between PDE gamma and the catalytic subunits.

3',5'-Cyclic-GMP Phosphodiesterases↗