Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3

Flajolet, Marc; Rakhilin, Sergey; Wang, Hong; Starkova, Natalia; Nuangchamnong, Nina; Nairn, Angus C.; Greengard, Paul

doi:10.1073/pnas.2136600100

Cited by 64 publications

(61 citation statements)

References 35 publications

(38 reference statements)

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“…Furthermore, none of the four major PPs is able to remove the phosphogroups completely from the I-II loop. Such differences are not attributable to the distinct intrinsic activities of these PPs and are similar to results by recent reports of PP2c and other PPs on other proteins (Desdouits et al, 1998;Flajolet et al, 2003). One likely scenario is that the phospho-groups on the channel I-II and II-III loops are nonhomogenous to these PPs, and not all of them can be accessed and/or dephosphorylated.…”

Section: Discussionsupporting

confidence: 86%

“…Consequently, the functional roles of PP2cs in the brain remain vir-tually unknown. Only a few neuronal substrates for PP2cs have been identified, among which are CaMKII (calcium/calmodulindependent protein kinase II), mGluR3 (metabotropic glutamate receptor subtype 3) and DARPP-32 (dopamine-and cAMPregulated phosphoprotein of 32 kDa) (Fukunaga et al, 1993;Desdouits et al, 1998;Flajolet et al, 2003). However, these results are based primarily on in vitro biochemical assays and have not yet been confirmed in vivo.…”

Section: Introductionmentioning

confidence: 99%

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A Protein Phosphatase 2cα-Ca²⁺Channel Complex for Dephosphorylation of Neuronal Ca²⁺Channels Phosphorylated by Protein Kinase C

Wang²,

Lai³

et al. 2005

J. Neurosci.

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Phosphorylation and dephosphorylation are primary means for rapid regulation of a variety of neuronal functions, such as membrane excitability, neurotransmitter release, and gene expression. Voltage-gated Ca 2ϩ channels are targets for phosphorylation by a variety of second messengers through activation of different types of protein kinases (PKs). Protein phosphatases (PPs), like PKs, are equally important in regulating Ca 2ϩ channels in neurons. However, much less is understood about whether and how a particular type of PP contributes to regulating neuronal Ca 2ϩ channel activities. This is primarily because of the lack of specific inhibitors/activators for different types of PPs, particularly the PP2c family. The functional roles of PP2c and its substrates in the brain remain virtually unknown. During our yeast two-hybrid screening, PP2c␣ was pulled out by both N-and P/Q-type Ca 2ϩ channel C termini. This raised the possibility that PP2c␣ might be associated with voltage-gated Ca 2ϩ channels for regulation of the Ca 2ϩ channel activity. Biochemical studies show that PP2c␣ binds directly to neuronal Ca 2ϩ channels forming a functional protein complex in vivo. PP2c␣, unlike PP1, PP2a and PP2b, is more effective in dephosphorylation of neuronal Ca 2ϩ channels after their phosphorylation by PKC. In hippocampal neurons, disruption of the PP2c␣-Ca 2ϩ channel interaction significantly enhances the response of Ca 2ϩ channels to modulation by PKC. Thus, the PP2c␣-Ca 2ϩ channel complex is responsible for rapid dephosphorylation of Ca 2ϩ channels and may contribute to regulation of synaptic transmission in neurons.

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Section: Discussionsupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

A Protein Phosphatase 2cα-Ca²⁺Channel Complex for Dephosphorylation of Neuronal Ca²⁺Channels Phosphorylated by Protein Kinase C

Wang²,

Lai³

et al. 2005

J. Neurosci.

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“…No screens were successful with full-length CaMKI because of poor mating efficiency. A random/oligonucleotide-dT rat brain cDNA library cloned in fusion with the Gal4 activation domain of pACT2 vector was used for all screens (Flajolet et al, 2003).…”

Section: Methodsmentioning

confidence: 99%

A Calcium- and Calmodulin-Dependent Kinase Iα/Microtubule Affinity Regulating Kinase 2 Signaling Cascade Mediates Calcium-Dependent Neurite Outgrowth

Uboha¹,

Flajolet²,

Nairn³

et al. 2007

J. Neurosci.

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Calcium is a critical regulator of neuronal differentiation and neurite outgrowth during development, as well as synaptic plasticity in adulthood. Calcium-and calmodulin-dependent kinase I (CaMKI) can regulate neurite outgrowth; however, the signal transduction cascades that lead to its physiological effects have not yet been elucidated. CaMKI␣ was therefore used as bait in a yeast two-hybrid assay and microtubule affinity regulating kinase 2 (MARK2)/Par-1b was identified as an interacting partner of CaMKI in three independent screens. The interaction between CaMKI and MARK2 was confirmed in vitro and in vivo by coimmunoprecipitation. CaMKI binds MARK2 within its kinase domain, but only if it is activated by calcium and calmodulin. Expression of CaMKI and MARK2 in Neuro-2A (N2a) cells and in primary hippocampal neurons promotes neurite outgrowth, an effect dependent on the catalytic activities of these enzymes. In addition, decreasing MARK2 activity blocks the ability of the calcium ionophore ionomycin to promote neurite outgrowth. Finally, CaMKI phosphorylates MARK2 on novel sites within its kinase domain. Mutation of these phosphorylation sites decreases both MARK2 kinase activity and its ability to promote neurite outgrowth. Interaction of MARK2 with CaMKI results in a novel, calciumdependent pathway that plays an important role in neuronal differentiation.

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“…The screens were performed as described (38), but using full-length CK2␣ and CK2␤ subunits fused to the GAL4 DNA-binding domain using a pAS2-derived bait vector (see SI Materials and Methods).…”

Section: Construction Of Expression Vectors and Rnaimentioning

confidence: 99%

“…For the in vitro experiments, transcribed and translated G␣ s or G␣olf were generated according to the Promega TNT kit protocol. Alternatively, striatal homogenates were prepared from female Sprague-Dawley rats as described (38).…”

Section: Construction Of Expression Vectors and Rnaimentioning

confidence: 99%

CK2 negatively regulates Gα _s signaling

Rebholz

Nishi²,

Liebscher³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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We present evidence, using biochemical and cellular approaches, that the kinase, CK2, negatively controls signaling via G␣ s (or G␣olf) coupled to dopamine D1 and adenosine A2A receptors. Pharmacological inhibition of CK2 or CK2 knockdown by RNAi lead to elevated cAMP levels in dopamine D1 receptor-activated neuroblastoma cells. Phosphorylation levels of protein kinase A substrates were increased in the presence of CK2 inhibitors in mouse striatal slices. The effect of D1 receptor and A2A receptor agonists on the phosphorylation of protein kinase A sites was potentiated upon CK2 inhibition. Furthermore, in cell lines, we observed that reduction in CK2 activity, pharmacologically or genetically, reduced the amount of D1 receptor that was internalized in response to dopamine. Finally, the ␤ subunit of CK2 was found to interact specifically with the G␣ s subunit through protein interaction analyses. Thus CK2 can inhibit G protein-coupled receptor action by enabling faster receptor internalization, possibly through a direct association with G␣ s.dopamine 1 receptor ͉ GPCR ͉ striatum ͉ casein kinase 2 ͉ adenosine A2A receptor G protein coupled receptors (GPCRs) make up the largest protein family in the human genome and consist of approximately 1,000 members. These receptors mediate the biological actions of neurotransmitters, hormones, pheromones, light, and calcium through the activation of 1 or more of the 4 G protein families G␣ i/o , G␣ q/11 , G␣ s , and G␣ 12/13 . Roughly 15% of 170 well-studied non-olfactory GPCRs signal via G␣ s which activates adenylyl cyclase (1).GPCR cell surface expression and coupling to G-proteins are regulated by phosphorylation of their third intracellular loop and/or their C-terminal region by various Ser-Thr kinases, such as G protein-coupled receptor kinases (GRKs1-7) (2). Binding of arrestins to GRK-phosphorylated receptors results in uncoupling of the receptor and in desensitization of the response (2). The phosphorylated GPCR/arrestin complex is then ready for endocytosis and is targeted to clathrin-coated pits, internalized, and either dephosphorylated before recycling to the plasma membrane, or targeted to lysosomes for degradation (3). In addition to the GRKs, second messenger-dependent protein kinases, such as protein kinase A (PKA) and protein kinase C (PKC), are involved in GPCR desensitization (3). Kinases including CK1␣ have been shown to phosphorylate other GPCRs, such as the M3-muscarinic receptor (4).In the current study, we sought to examine the possible role of additional protein kinases in GPCR signaling. In particular, we wished to examine the regulation of dopamine-coupled GPCRs in neurons. Dopamine signaling is of major importance for the biology of the striatum and is altered in various neurological and psychiatric diseases such as Parkinson's disease (PD), schizophrenia, attention deficit hyperactivity disorder (ADHD), and drug abuse (5). The dopamine D1 and D5 receptors signal via G␣ olf (a G␣ s subtype of G protein) to activate adenylyl cyclase, whereas D2, D...

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Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3

Cited by 64 publications

References 35 publications

A Protein Phosphatase 2cα-Ca²⁺Channel Complex for Dephosphorylation of Neuronal Ca²⁺Channels Phosphorylated by Protein Kinase C

A Protein Phosphatase 2cα-Ca²⁺Channel Complex for Dephosphorylation of Neuronal Ca²⁺Channels Phosphorylated by Protein Kinase C

A Calcium- and Calmodulin-Dependent Kinase Iα/Microtubule Affinity Regulating Kinase 2 Signaling Cascade Mediates Calcium-Dependent Neurite Outgrowth

CK2 negatively regulates Gα _s signaling

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Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3

Cited by 64 publications

References 35 publications

A Protein Phosphatase 2cα-Ca2+Channel Complex for Dephosphorylation of Neuronal Ca2+Channels Phosphorylated by Protein Kinase C

A Protein Phosphatase 2cα-Ca2+Channel Complex for Dephosphorylation of Neuronal Ca2+Channels Phosphorylated by Protein Kinase C

A Calcium- and Calmodulin-Dependent Kinase Iα/Microtubule Affinity Regulating Kinase 2 Signaling Cascade Mediates Calcium-Dependent Neurite Outgrowth

CK2 negatively regulates Gα s signaling

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A Protein Phosphatase 2cα-Ca²⁺Channel Complex for Dephosphorylation of Neuronal Ca²⁺Channels Phosphorylated by Protein Kinase C

A Protein Phosphatase 2cα-Ca²⁺Channel Complex for Dephosphorylation of Neuronal Ca²⁺Channels Phosphorylated by Protein Kinase C

CK2 negatively regulates Gα _s signaling