Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase.

Willardson, Barry M.; Wilkins, Jon F.; Yoshida, Tatsuro; Bitensky, Mark W.

doi:10.1073/pnas.93.4.1475

Cited by 55 publications

(63 citation statements)

References 38 publications

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“…The insensitivity to BAPTA-AM indicates that CaMKII is not involved in the phosphorylation of Ser-73 in intact retina despite its ability to phosphorylate Ser-73 in vitro (16). Moreover, this result shows that a Ca 2ϩ /calmodulin-dependent adenylyl cyclase does not make Ser-73 phosphorylation sensitive to Ca 2ϩ under these conditions, contrary to what was suggested previously from phosphorylation experiments using rod outer segment preparations (35).…”

Section: Effects Of Light and Ca 2ϩ On Phosphorylation Of Pdc Atcontrasting

confidence: 56%

“…As a result, Pdc only binds G t ␤␥ in the light. Data from the Pdc knock-out mouse show that the binding of Pdc to G t ␤␥ is not a mechanism of desensitization of short term light responses as was proposed previously (35,39) because there was no change in these responses in the absence of Pdc (23). On the other hand, the translocation of G t ␤␥ from the outer segment to the inner segment during light adaptation, which leads to desensitization during long term light responses, was disrupted in the absence of Pdc (23).…”

Section: Light-dependent Regulation Of Ser-54 and Ser-73 Phosphorylatmentioning

confidence: 68%

“…The PKA-phosphorylated Pdc had the following distribution of phosphorylated species: no phosphates (3%), one (5%), two (12%), three (44%), or four (36%). Ser-54 and Ser-73 harbor the consensus sites for phosphorylation by CaMKII and PKA, respectively, and are readily phosphorylated by these kinases (13,16,35), therefore they would be the first residues phosphorylated. As a result, Ser-54 and Ser-73 would be phosphorylated in all species containing one or more phosphates.…”

Section: Methodsmentioning

confidence: 99%

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Site-specific Phosphorylation of Phosducin in Intact Retina

Lee

Thulin

Willardson

2004

Journal of Biological Chemistry

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Phosducin (Pdc) is a G protein ␤␥ dimer (G␤␥) binding protein, highly expressed in retinal photoreceptor and pineal cells, yet whose physiological role remains elusive. Light controls the phosphorylation of Pdc in a cAMP and Ca 2؉ -dependent manner, and phosphorylation in turn regulates the binding of Pdc to G t ␤␥ or 14-3-3 proteins in vitro. To directly examine the phosphorylation of Pdc in intact retina, we prepared antibodies specific to the three principal phosphorylation sites (Ser-54, Ser-73, and Ser-106) and measured the kinetics of phosphorylation/dephosphorylation during light/dark adaptation and the subsequent effects on G t ␤␥ binding. Ser-54 phosphorylation increased slowly (t1 ⁄2 ϳ 90 min) during dark adaptation to ϳ70% phosphorylated and decreased rapidly (t1 ⁄2 ϳ 2 min) during light adaptation to less than 20% phosphorylated. Ser-73 phosphorylation increased much faster during dark adaptation (t1 ⁄2 ϳ 3 min) to ϳ50% phosphorylated and decreased more slowly during light adaptation (t1 ⁄2 ϳ 9 min) to less than 20% phosphorylated. The Ca 2؉ chelator BAPTA-AM blocked Ser-54 phosphorylation during dark adaptation but had no effect on Ser-73 phosphorylation. In contrast, Ser-106 was not phosphorylated in either the light or dark. Importantly, G␤␥ binding to Pdc was enhanced by Ca 2؉ chelation and the binding kinetics closely paralleled those of Ser-54 dephosphorylation, indicating that Ser-54 phosphorylation controls G t ␤␥ binding in vivo. These results suggest a pivotal role of Ser-54 and Ser-73 phosphorylation in determining the interactions of Pdc with its binding partners, G t ␤␥ and 14-3-3 protein, which may regulate the light-dependent translocation of the photoreceptor G protein.G protein-coupled receptors mediate cellular responses to a wide variety of signals including hormones, neurotransmitters, odorants, chemoattractants, and photons of light. The conversion of light into a neural response by the photoreceptor cells of the vertebrate retina is initiated by a canonical G protein signaling pathway involving the receptor rhodopsin, the photoreceptor G protein, transducin (G t ), 1 cGMP phosphodiesterase, and cGMP-gated cation channels (1). Closure of the channels in response to light causes a hyperpolarization of the photoreceptor cell membrane, which triggers the neural response. In addition, channel closure also results in a decrease in Ca 2ϩ in the photoreceptor cell, which orchestrates a number of molecular events that lead to a decrease in sensitivity of the photoreceptor to light, a process termed light adaptation (2).The role of the G t ␣ subunit in activation of the phosphodiesterase has been extensively studied (3-5), as has regulation of its GTPase activity by RGS-9 (6 -8). In contrast, the only known function of the G t ␤␥ subunit complex is to mediate the interaction of G t ␣ with rhodopsin, yet in other G protein signaling systems G␤␥ plays significant roles in downstream effector activation (9). Besides G t ␣, the only other reported binding partner for G t ␤␥ in photoreceptor c...

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Section: Effects Of Light and Ca 2ϩ On Phosphorylation Of Pdc Atcontrasting

confidence: 56%

Section: Light-dependent Regulation Of Ser-54 and Ser-73 Phosphorylatmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

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Site-specific Phosphorylation of Phosducin in Intact Retina

Lee

Thulin

Willardson

2004

Journal of Biological Chemistry

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“…Cytosolic Ca 2+ levels fall upon illumination of photoreceptors. This drop in Ca 2+ levels affects the activity of a Ca 2+ /calmodulin-dependent adenylyl cyclase, dropping the levels of cAMP, which, in turn, decreases the level of net cAMP-dependent phosphorylation of phosducin (66). The drop in cytosolic Ca 2+ levels also affects the activity of the Ca 2+ -dependent kinase, decreasing its activity and the net phosphorylation state of phosducin.…”

Section: Discussionmentioning

confidence: 99%

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

et al. 2002

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In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B′ subunit, B56epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B′ subunit (sequenced to be B56alpha) or with the BR regulatory subunit have no phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (∼20 ng/mouse retina) and 9 times more of the C subunit (∼4 ng/mouse retina) than of the B56epsilon subunit (∼0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56epsilon subunit, greatly prefers phosphorylated phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic phosducin and membrane-bound rhodopsin, by the same enzyme.

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“…Moreover, its ability to inhibit G protein signaling was reversed when Pdc was phosphorylated by PKA [13,14]. These findings led to the hypothesis that Pdc could be involved in light-adaptation in photoreceptor cells through a feedback inhibition cycle of light-dependent phosphorylation events [32]. The logic of this hypothesis was as follows.…”

Section: Early Observations -The Gβγ Sequestration Hypothesismentioning

confidence: 99%

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Willardson

Howlett

2007

Cellular Signalling

100

101

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Members of the phosducin gene family were initially proposed to act as down-regulators of G protein signaling by binding G protein βγ dimers (Gβγ) and inhibiting their ability to interact with G protein α subunits (Gα) and effectors. However, recent findings have overturned this hypothesis by showing that most members of the phosducin family act as cochaperones with the cytosolic chaperonin complex (CCT) to assist in the folding of a variety of proteins from their nascent polypeptides. In fact rather than inhibiting G protein pathways, phosducin-like protein 1 (PhLP1) has been shown to be essential for G protein signaling by catalyzing the folding and assembly of the Gβγ dimer. PhLP2 and PhLP3 have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tubulin. Phosducin itself is the only family member that does not participate with CCT in protein folding, but it is believed to have a specific role in visual signal transduction to chaperone Gβγ subunits as they translocate to and from the outer and inner segments of photoreceptor cells during light-adaptation.

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Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase.

Cited by 55 publications

References 38 publications

Site-specific Phosphorylation of Phosducin in Intact Retina

Site-specific Phosphorylation of Phosducin in Intact Retina

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

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