Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina

Lee, Rehwa H.; Brown, B. M.; Lolley, Richard N.

doi:10.1021/bi00304a014

Cited by 129 publications

(78 citation statements)

References 23 publications

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“…Several previous studies suggested that Ser-73 is phosphorylated in photoreceptor cells in the dark by PKA as a result of an increase in cAMP (12)(13)(14)(15). The results reported here confirm those studies and also show that the phosphorylation in the dark is quite rapid with a t1 ⁄2 of ϳ3 min, consistent with a robust PKA activity reported previously in photoreceptor cells (12)(13)(14)(15)(16).…”

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

confidence: 92%

“…Besides G t ␣, the only other reported binding partner for G t ␤␥ in photoreceptor cells is phosducin (Pdc), a 28-kDa phosphoprotein expressed at high levels in both photoreceptor cells and pinealocytes (10,11). Pdc is phosphorylated in dark-adapted photoreceptors and dephosphorylated in response to light (12). Pdc harbors consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) at Ser-73 and Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) at Ser-54, and the results of several studies indicate that PKA and CaMKII are the relevant kinases for Pdc phosphorylation (13)(14)(15)(16).…”

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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: Light-dependent Regulation Of Ser-54 and Ser-73 Phosphorylatsupporting

confidence: 92%

mentioning

confidence: 99%

Site-specific Phosphorylation of Phosducin in Intact Retina

Lee

Thulin

Willardson

2004

Journal of Biological Chemistry

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“…In strong light, when maximum modulation is most desirable so that the cascade is not saturated, the net dephosphorylated state of phosducin is ensured by several synergistic mechanisms. The fall in photoreceptor cGMP levels upon illumination results in decreased levels of phosphorylated phosducin, in part because the PKA enzyme that phosphorylates phosducin is also sensitive to cGMP levels (22). Cytosolic Ca 2+ levels fall upon illumination of photoreceptors.…”

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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“…Pdc was first discovered as a retinal phospho-protein that was phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and subsequently dephosphorylated upon exposure to light [31]. Interest in Pdc increased significantly when it was shown to co-purify from retinal extracts with the βγ subunit complex of the retinal G protein, transducin (G t βγ) [11].…”

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

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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Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina

Cited by 129 publications

References 23 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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