Synucleins Are a Novel Class of Substrates for G Protein-coupled Receptor Kinases
2000; Elsevier BV; Volume: 275; Issue: 34 Linguagem: Inglês
10.1074/jbc.m003542200
ISSN1083-351X
AutoresAlexey Pronin, Andrew J. Morris, Andrei Surguchov, Jeffrey Benovic,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoG protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (α, β, γ, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. α-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the α and β isoforms, whereas GRK5 prefers α-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gβγ subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein. G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (α, β, γ, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. α-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the α and β isoforms, whereas GRK5 prefers α-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gβγ subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein. G protein-coupled receptor kinase calmodulin-dependent protein kinase II casein kinase G protein-coupled receptor phospholipase D polymerase chain reaction phosphatidylcholine fast protein liquid chromatography polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine G protein-coupled receptor kinases (GRKs)1 are involved in the regulation of G protein-coupled receptor (GPCR) signaling (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (232) Google Scholar, 2Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar). GRKs specifically recognize and phosphorylate agonist-occupied GPCRs. Receptor phosphorylation and subsequent binding of another protein, arrestin, uncouples activated receptor from G protein. These events can also promote receptor endocytosis. Internalized receptors are then either dephosphorylated and recycled back to the cell surface or targeted to lysosomes for degradation. The seven mammalian GRKs that have been identified can be divided into three subfamilies based on their overall structural organization and homology: GRK1 (rhodopsin kinase) and GRK7; GRK2 (βARK1) and GRK3 (βARK2); and GRK4, GRK5, and GRK6. Common features shared by the GRKs include a centrally localized catalytic domain of ∼270 amino acids, an N-terminal domain of ∼190 amino acids that has been implicated in receptor interaction and GRK regulation, and a variable length C-terminal domain of 105–233 amino acids that is involved in phospholipid association.Until recently, GPCRs were considered to be the only natural substrates for GRKs. Common protein kinase substrates, such as casein, phosvitin, and synthetic peptides are relatively poor substrates for GRKs. Even inactive GPCRs are not phosphorylated well. These observations suggest that GRKs might be highly specialized kinases, which phosphorylate only activated receptors. However, several laboratories recently demonstrated that GRKs can also bind and effectively phosphorylate the cytoskeletal protein tubulin (3Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.G. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 4Carman C.V. Som T. Kim C.M. Benovic J.L. J. Biol. Chem. 1998; 273: 20308-20316Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 5Haga K. Ogawa H. Haga T. Murofushi H. Eur. J. Biochem. 1998; 255: 363-368Crossref PubMed Scopus (51) Google Scholar). Interestingly, GRK-mediated phosphorylation of tubulin appears to be stimulated by β2-adrenergic receptor activation (3Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.G. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In addition, GRK6 appears to selectively phosphorylate the Na+/H+ exchanger regulatory factor via a PDZ domain-mediated interaction (6Hall R.A. Spurney R.F. Premont R.T. Rahman N. Blitzer J.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 24328-24334Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Although the physiological role for GRK-mediated phosphorylation of tubulin and the Na+/H+ exchanger regulatory factor is unclear at present, these examples suggest that GRKs may have a broader substrate selectivity than previously assumed and thus play a wider role in signaling than previously appreciated.In this study, we explored the possibility that additional nonreceptor GRK substrates exist. In our search for such substrates, we identified synucleins as a novel family of GRK substrates. Synucleins have been linked to the development of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, and may be involved in regulating intracellular vesicular trafficking (7Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (267) Google Scholar). We identified the site phosphorylated by GRKs in α-synuclein and demonstrated that this phosphorylation inhibits the ability of α-synuclein to interact with phospholipids and results in reduced inhibition of phospholipase D2 (PLD2).DISCUSSIONA significant role of GRKs in the regulation of GPCR-mediated signaling is well established (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (232) Google Scholar, 2Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar). Many experiments, which include gene-targeted knockouts and transgenic protein overexpression, demonstrated the importance of GRK-mediated phosphorylation in receptor desensitization. However, our understanding of GRK function is far from complete, and research continues to uncover novel ways of GRK regulation as well as new functions for these kinases. For example, the identification of an RGS homology domain in GRKs (22Siderovski D.P. Hessel A. Chung S. Mak T.W. Tyers M. Curr. Biol. 1996; 6: 211-212Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) led to the discovery that GRK2 and GRK3 can specifically interact with activated αq/11 and inhibit αq/11-mediated signaling (23Carman C.V. Parent J.-L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). This finding demonstrates that GRKs might be able to regulate signaling in a manner that is independent of their enzymatic activity.Other recent studies have shown that GRK phosphorylation is not limited to activated receptors as substrates. GRKs can also efficiently phosphorylate nonreceptor protein substrates, such as Na+/H+ exchanger regulatory factor (6Hall R.A. Spurney R.F. Premont R.T. Rahman N. Blitzer J.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 24328-24334Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and tubulin (3Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.G. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 4Carman C.V. Som T. Kim C.M. Benovic J.L. J. Biol. Chem. 1998; 273: 20308-20316Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 5Haga K. Ogawa H. Haga T. Murofushi H. Eur. J. Biochem. 1998; 255: 363-368Crossref PubMed Scopus (51) Google Scholar). Although the role for this phosphorylation is undetermined, phosphorylation of tubulin suggests that GRKs might be involved in regulating microtubule dynamics and cytoskeletal reorganization. The cytoskeleton plays an important role in assembly of signaling networks and is also involved in such processes as endocytosis and vesicular trafficking. It is possible that GRK-mediated tubulin phosphorylation during a signaling event activates rearrangement of microtubules, thus contributing to receptor internalization, recycling, or degradation. It is also possible that GRKs might be involved in the regulation of signaling and cytoskeleton dynamics by phosphorylating additional protein substrates.In our search for novel nonreceptor GRK substrates, we identified α-synuclein as a protein phosphorylated by GRKs in a brain extract. α-Synuclein belongs to a family of small proteins (127–140 amino acids), which currently includes four members (α-, β-, and γ-synucleins and synoretin) (7Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (267) Google Scholar, 10Surguchov A. Surguchova I. Solessio E. Baehr W. Mol. Cell. Neurosci. 1999; 13: 95-103Crossref PubMed Scopus (80) Google Scholar). Synucleins are expressed at high levels in the brain and at lower levels in many tissues and common cell lines such as 293 and COS. α-Synuclein has been linked to the development of neurodegenerative disease. For example, a fragment of α-synuclein is a component of senile plaques of Alzheimer's disease patients. α-Synuclein is also found within Lewy bodies inside neurons of Parkinson's disease patients. Indeed, two independent autosomal mutations (A53T and A30P) in the α-synuclein gene have been linked to the early onset inherited forms of Parkinson's disease in two families (24Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6513) Google Scholar, 25Krüger R. Kuhn W. Müller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3263) Google Scholar). Moreover, overexpression of human α-synuclein in transgenic mice resulted in progressive accumulation of Lewy body-like structures in neurons and a loss of dopaminergic terminals in the basal ganglia (26Masliah E. Rockenstein E. Veinbergs I. Mallory M. Hashimoto M. Takeda A. Sagara Y. Sisk A. Mucke L. Science. 2000; 287: 1265-1269Crossref PubMed Scopus (1538) Google Scholar). Another member of the synuclein family, γ-synuclein, may play a role in the etiology of breast cancer (27Ji H. Liu Y.E. Jia T. Wang M. Liu J. Xiao G. Joseph B.K. Rosen C. Shi Y.E. Cancer Res. 1997; 57: 759-764PubMed Google Scholar). Whereas the biological role of synucleins is not yet defined, recent studies have begun to shed some light on their functions. For example, synucleins have the ability to form fibrils in vitro (28Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1242) Google Scholar). The accelerated fibril formation by mutant synucleins may contribute to the development of neurodegenerative diseases. The intracellular localization of synucleins is also not well defined. Although some studies have shown that synucleins are cytosolic proteins, others have demonstrated that synucleins are associated with synaptic vesicles (7Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (267) Google Scholar) and other membranes (29McLean P.J. Kawamata H. Ribich S. Hyman B.T. J. Biol. Chem. 2000; 275: 8812-8816Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). α-Synuclein was shown to interact with phospholipids. This interaction is facilitated mainly by a conserved N-terminal domain (amino acid residues 1–94), which changes its structure from “unfolded” to α-helical upon binding to lipids (21Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1213) Google Scholar). Because of synuclein's ability to interact with lipids and their association with synaptic vesicles, it has been suggested that synucleins might be involved in intracellular vesicular trafficking (7Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (267) Google Scholar).Recent studies have identified some proteins that can interact with synucleins. Synucleins can bind to 14-3-3 proteins, protein kinase C, BAD, and Erk (30Ostrerova N. Petrucelli L. Farrer M. Mehta N. Choi P. Hardy J. Wolozin B. J. Neurosci. 1999; 19: 5782-5791Crossref PubMed Google Scholar). They are also potent inhibitors of PLD2 in vitro (16Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (378) Google Scholar), whereas overexpression of synoretin activates Elk-1 transcription factor (10Surguchov A. Surguchova I. Solessio E. Baehr W. Mol. Cell. Neurosci. 1999; 13: 95-103Crossref PubMed Scopus (80) Google Scholar). It has been also suggested that synucleins can be regulated by phosphorylation (7Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (267) Google Scholar, 16Jenco J.M. Rawlingson A. Daniels B. Morris A.J. Biochemistry. 1998; 37: 4901-4909Crossref PubMed Scopus (378) Google Scholar), since bovine β-synuclein occurs as a phosphoprotein and can be phosphorylated in vitro by CaMKII (17Nakajo S. Tsukada K. Omata K. Nakamura Y. Nakaya K. Eur. J. Biochem. 1993; 217: 1057-1063Crossref PubMed Scopus (148) Google Scholar). Recently, it has been shown that α-synuclein is phosphorylated in cells and that phosphosynuclein is rapidly dephosphorylated by okadaic acid-sensitive protein phosphatases (18Okochi M. Walter J. Koyama A. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P. Haas C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). This study also demonstrated that the casein kinases CK1 and CK2 can phosphorylate α-synuclein in vitro. However, whether these kinases phosphorylate other synuclein subtypes and what regulates phosphorylation and the consequences of synuclein phosphorylation have not been determined.Here we demonstrated that all four synucleins are substrates for GRKsin vitro. All tested GRKs can phosphorylate synucleins, albeit with different efficiency. α-Synuclein appears to be the best substrate for all GRKs, whereas γ-synuclein and synoretin are phosphorylated significantly slower. Of all tested kinases, GRK2 phosphorylates β-synuclein most efficiently. Other kinases can also phosphorylate synucleins. As has been reported, both CK1 and CK2 phosphorylate α-synuclein. In addition, CK2 can effectively phosphorylate γ-synuclein but not β-synuclein or synoretin. CaMKII phosphorylates γ-synuclein best, although not very efficiently. Co-expression of α-synuclein with GRKs in COS-1 cells demonstrated that GRK2 and GRK5 can also phosphorylate this protein in cells.We determined that synuclein phosphorylation by GRKs can be regulated by some of the factors that are also known to regulate receptor phosphorylation. Synuclein phosphorylation by all GRKs is enhanced by phospholipids. For GRK2, this effect is dependent on the presence of Gβγ. Because Gβγ is believed to be released after receptor activation, it is plausible that activation of receptors by extracellular stimuli may lead to synuclein phosphorylation via activated GRK2. Because both GRK and synuclein can bind to liposomes, the mechanism for the lipid-mediated increase in synuclein phosphorylation is unclear. One possibility is that synucleins undergo a conformational change when they bind to phospholipids, making them a better substrate for GRKs. Another possibility is that phospholipids directly activate the catalytic activity of GRKs. The latter seems more likely, since the EC50 (0.04 mg/ml phospholipid) for GRK5-mediated α-synuclein phosphorylation by liposomes closely matches the EC50 for GRK5 binding to liposomes (31Pronin A.N. Carman C.V. Benovic J.L. J. Biol. Chem. 1998; 273: 31510-31518Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and is significantly lower than the EC50 for α-synuclein binding to phospholipids (Fig. 7 A). Interestingly, liposomes also enhance the phosphorylation of α-synuclein by CK2. However, this effect is not universal; phospholipids have no effect on α-synuclein phosphorylation by CK1 or γ-synuclein phosphorylation by CK2.Surprisingly, phosphorylation of α-synuclein by GRK5 is also activated by Ca2+/calmodulin, which is known to potently inhibit GRK5-mediated receptor phosphorylation (8Pronin A.N. Satpaev D.K. Slepak V.Z. Benovic J.L. J. Biol. Chem. 1997; 272: 18273-18280Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Chuang T.T. Paolucci L. De Blasi A. J. Biol. Chem. 1996; 271: 28691-28696Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This finding suggests that regulation of GRK5 by calmodulin may be more complex than previously thought. Calmodulin may not only regulate the catalytic activity of GRK5, but it may also change the substrate specificity of the kinase. Calmodulin-dependent activation of phosphorylation appears to be GRK and synuclein subtype-specific. Synuclein phosphorylation by GRK2 was weakly inhibited in the presence of calmodulin, whereas GRK5 phosphorylation of γ-synuclein was virtually unaffected. γ-Synuclein, on the other hand, is phosphorylated by CaMKII in a calmodulin-dependent manner. Thus, calmodulin activation in cells may lead to phosphorylation of different synuclein subtypes depending on which kinase is expressed in a particular cell type. Assuming that different synucleins have distinct functions, calmodulin-dependent synuclein phosphorylation may have different functional effects.GRKs appear to phosphorylate a single site in the C-tail of synucleins (Ser129 in α-, Ser118 in β-, and Ser124 in γ-synuclein, respectively). In α-synuclein, this is the same residue that is endogenously phosphorylated in 293 cells (18Okochi M. Walter J. Koyama A. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P. Haas C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). In some synuclein subtypes, these sites can also be phosphorylated by other kinases, such as CK1, CK2, or CaMKII. C-terminal regions of synucleins vary significantly from each other. However, the serine residue phosphorylated by GRKs in synucleins appears to be rather conserved (Fig. 5 B). In all synucleins, this residue is surrounded by several acidic residues. Interestingly, earlier studies with peptide substrates suggested that GRKs may prefer different environments for the residues they use as substrates; GRK2 prefers an acidic environment, whereas GRK5 prefers an uncharged or possibly basic environment (13Kunapuli P. Onorato J.J. Hosey M.M. Benovic J.L. J. Biol. Chem. 1994; 269: 1099-1105Abstract Full Text PDF PubMed Google Scholar). Contrary to this notion, however, GRK5 phosphorylates α-synuclein as efficiently as GRK2. Nevertheless, both the surrounding residues and more distant parts of the molecule undoubtedly play an important role in determining substrate specificity of the kinase. For example, β-synuclein, which is most closely related to α-synuclein and which is a good substrate for GRK2, is a very poor substrate for CK2. Interestingly, phospholipids also stimulate GRK5 phosphorylation of residues in γ-synuclein and synoretin that are distinct from the residue phosphorylated by other kinases (Ser124). It seems likely that these additional phosphorylation sites are exposed when synucleins undergo a conformational change upon binding to liposomes. The identity and function of these sites is under investigation.Although the normal function of synuclein is not clear, some data indicate that synucleins may play a role in the regulation of intracellular vesicular trafficking and signaling. A recent report suggests that α-synuclein may be involved in desensitization of dopamine signaling (32Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.-H. Castillo P.E. Shinsky N. Verdugo J.M.G. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar). Mice lacking α-synuclein display a standard pattern of dopamine discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli, supporting the idea that α-synuclein is a negative regulator of dopamine neurotransmission, possibly by modulating the refilling rate of the readily releasable synaptic vesicles. This modulation may involve either a direct interaction of synuclein with vesicles or regulation of an enzyme activity such as PLD2. Here we demonstrated that GRK-mediated phosphorylation inhibits both synuclein's ability to interact with lipids and modulate the activity of PLD2. It is likely that the reduction in synuclein's ability to inhibit PLD2 is due to the reduced affinity of phosphorylated synuclein for phospholipids. One can speculate about the possible role of GRK-mediated synuclein phosphorylation in signal regulation. PLD catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid and diacylglycerol. Phosphatidic acid has been shown to stimulate vesicle formation (33Jones D. Morgan C. Cockcroft S. Biochim. Biophys. Acta. 1999; 1439: 229-244Crossref PubMed Scopus (167) Google Scholar). Increased PLD2 activity also causes rearrangement of cortical actin cytoskeleton (15Colley W.C. Sung T.-C. Roll R. Jenco J. Hammond S.M. Altshuller E. Bar-Sagi D. Morris A.J. Frohman M.A. Curr. Biol. 1997; 7: 191-201Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). Both of these processes are involved in receptor endocytosis and/or recycling. Thus, activated receptors, which in turn can activate GRKs, get phosphorylated and targeted for endocytosis. Activated GRKs can also phosphorylate synuclein, thus releasing inhibition of PLD2. Activation of PLD2 causes rearrangement of actin cytoskeleton and vesicle formation aiding in receptor endocytosis and/or recycling.An additional mechanism by which synuclein phosphorylation might be linked to regulation of cytoskeleton comes from a recent report that α-synuclein can bind to tau protein and promote tau phosphorylation by protein kinase A (34Jensen P.H. Hager H. Nielsen M.S. Højrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Tau is involved in the regulation of microtubule dynamics. Interestingly, the synuclein residue that is phosphorylated by GRKs (and CKs) lies inside the region (residues 87–140) that interacts with tau. Thus, it is possible that synuclein phosphorylation affects its interaction with tau and alters the dynamics of microtubule assembly. GRK phosphorylation of tubulin may also directly affect tubulin cytoskeleton formation. The findings that GRKs can directly interact with cytoskeletal proteins such as tubulin and actin (3Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.G. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 4Carman C.V. Som T. Kim C.M. Benovic J.L. J. Biol. Chem. 1998; 273: 20308-20316Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 5Haga K. Ogawa H. Haga T. Murofushi H. Eur. J. Biochem. 1998; 255: 363-368Crossref PubMed Scopus (51) Google Scholar) and the identification of synucleins as GRK substrates suggest an important role for GRKs in the regulation of cytoskeletal dynamics. Identification of novel GRK substrates may help us discover yet unknown cellular functions for these kinases. G protein-coupled receptor kinases (GRKs)1 are involved in the regulation of G protein-coupled receptor (GPCR) signaling (1Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (232) Google Scholar, 2Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar). GRKs specifically recognize and phosphorylate agonist-occupied GPCRs. Receptor phosphorylation and subsequent binding of another protein, arrestin, uncouples activated receptor from G protein. These events can also promote receptor endocytosis. Internalized receptors are then either dephosphorylated and recycled back to the cell surface or targeted to lysosomes for degradation. The seven mammalian GRKs that have been identified can be divided into three subfamilies based on their overall structural organization and homology: GRK1 (rhodopsin kinase) and GRK7; GRK2 (βARK1) and GRK3 (βARK2); and GRK4, GRK5, and GRK6. Common features shared by the GRKs include a centrally localized catalytic domain of ∼270 amino acids, an N-terminal domain of ∼190 amino acids that has been implicated in receptor interaction and GRK regulation, and a variable length C-terminal domain of 105–233 amino acids that is involved in phospholipid association. Until recently, GPCRs were considered to be the only natural substrates for GRKs. Common protein kinase substrates, such as casein, phosvitin, and synthetic peptides are relatively poor substrates for GRKs. Even inactive GPCRs are not phosphorylated well. These observations suggest that GRKs might be highly specialized kinases, which phosphorylate only activated receptors. However, several laboratories recently demonstrated that GRKs can also bind and effectively phosphorylate the cytoskeletal protein tubulin (3Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.G. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 4Carman C.V. Som T. Kim C.M. Benovic J.L. J. Biol. Chem. 1998; 273: 20308-20316Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 5Haga K. Ogawa H. Haga T. Murofushi H. Eur. J. Biochem. 1998; 255: 363-368Crossref PubMed Scopus (51) Google Scholar). Interestingly, GRK-mediated phosphorylation of tubulin appears to be stimulated by β2-adrenergic receptor activation (3Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.G. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In addition, GRK6 appears to selectively phosphorylate the Na+/H+ exchanger regulatory factor via a PDZ domain-mediated interaction (6Hall R.A. Spurney R.F. Premont R.T. Rahman N. Blitzer J.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 24328-24334Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Although the physiological role for GRK-mediated phosphorylation of tubulin and the Na+/H+ exchanger regulatory factor is unclear at present, these examples suggest that GRKs may have a broader substrate selectivity than previously assumed and thus play a wider role in signaling than previously appreciated. In this study, we explored the possibility that additional nonreceptor GRK substrates exist. In our search for such substrates, we identified synucleins as a novel family of GRK substrates. Synucleins have been linked to the development of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, and may be involved in regulating intracellular vesicular traff
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