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J Cunnick

Publications and source records attributed to J Cunnick.

15 recordsLinked to original sources
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

Retinal cyclic-GMP phosphodiesterase gamma-subunit: identification of functional residues in the inhibitory region.

Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides. The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues # 24-45 and an inhibitory region for PDE alpha/beta within residues # 80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, peptides of PDE gamma # 63-87 and mutant peptides were synthesized and utilized in PDE inhibition assays. The following peptides exhibited a decreased ability to inhibit PDE alpha/beta: All were from PDE gamma # 63-87; PDE gamma Tyr 84----Gly, PDE gamma Phe 73----Gly and PDE gamma Gln 83----Gly.

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

Retinal cyclic-GMP phosphodiesterase gamma-subunit: use of mutant synthetic peptides to define function.

Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides (1). The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues #24-45. An inhibitory region for PDE alpha/beta is within residues #80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, mutants were synthesized and utilized in PDE inhibition assays. The following mutants exhibited a decreased ability to inhibit PDE alpha/beta: Tyr84----Gly; Arg24----Gly; and Arg33----Pro. Sequence comparisons with cone PDE gamma indicate that there is identity within these functional regions.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

Analysis of normal and rcdl Irish setter retinal proteins.

In both of the early onset systems, the rd mouse and the rcdl Irish Setter, early elevation of cyclic GMP may be the ultimate cause of accelerated photoreceptor degeneration. This would be consistent with the data utilizing in vitro systems in which retinal samples, in culture, undergo degeneration in response to constant exposure to high levels of this nucleotide. However, the ultimate cause of the elevated cyclic GMP in the rd mouse or in the rcdl Irish Setter still remains a mystery. It appears that all of the necessary proteins of the visual cascade are produced, although they are lost at different rates. The phosphodiesterase appears to be reduced faster than other proteins. This may, in turn, account for the elevation in cyclic GMP levels. The cause of this enhanced disappearance could reside in the phosphodiesterase protein itself, or in other more distal components. The alteration in rhodopsin reaction to the specific rhod-4 antisera suggests that this protein is not properly oriented in the disc membrane. Although this may or may not alter the visual cascade, it does suggest that these membranes are not identical to those of the normal dog retina. Future studies should focus on the individual functional activities of each component, on their structures, and on their proper assembly within the disc.

Amino Acid Sequence↗
PubMed1988

Rod/cone dysplasia in Irish setters. Presence of an altered rhodopsin.

On the basis of the amino acid sequence of bovine rhodopsin, a series of peptides from the C-terminus (Rhod-4 and Rhod-1) and external loops (Rhod-10) were synthesized. Rabbit antisera to these peptides recognize the rhodopsin molecule in whole retina from 8-week-old normal and affected rcdl (rod/cone-dysplasic) Irish setters (8- and 4-weeks-old). When the rhodopsin content was equalized by using a solid-phase radioimmunoassay, the reaction with anti-peptide antisera to the C-terminal octapeptide (residues 341-348) is severely decreased in the rcdl-dog retinas. The results of mixing experiments suggest that this is not due to proteolytic clipping of the rhodopsin C-terminus from the affected dogs. Treatment of retinas with 1.0 mM-NaF, a phosphatase inhibitor, or pretreatment with alkaline and acid phosphatases does alter the reaction of the rhodopsin with anti-rhodopsin antisera. This suggests that the decreased reaction of the affected rhodopsin with the anti-peptide antisera may partially result from differences in intrinsic rhodopsin phosphorylation. However, since the reaction of rcdl retinas cannot be restored to that of the normals, these results suggest that the rhodopsin molecule from the rcdl dogs may be structurally altered in other ways.

Alkaline Phosphatase↗
PubMed1986

Reduced rhodopsin phosphorylation during retinal dystrophy.

During inherited retinal dystrophy in Irish Setter dogs, decreased activity of cGMP phosphodiesterase (PDE) results in high cGMP levels and retinal degeneration (1-3). This defect could be in PDE itself, or in its interactions with other proteins of the rod outer segment. We report herein that when retinas from 8-week-old dogs were phosphorylated with gamma-32P-ATP, and separated on SDS-PAGE, phosphorylation of rd dog rhodopsin was reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Since rd-mediated inhibition was prevented by 1 mM NaF, the results suggest that the cause of reduced rd phosphorylation is increased phosphatase activity. Together, these results demonstrate that decreased phosphorylation of rhodopsin due to increased phosphatase activity is a fundamental biochemical change which may partially account for the degenerative process and loss of visual acuity during inherited retinal dystrophy.

Animals↗