Phospholipid-stimulated autophosphorylation activates the G protein ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Val. 269, No. 14, Issue of April 8,pp. 10209-10212, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication Phospholipid-stimulated Autophosphorylation Activates the G Protein-coupled Receptor Kinase GRK5* (Received for publication, February 1, 1994)

Priya Kunapuli, VsevolodV. Gurevich, and Jeffrey L. Benovicz From the Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

kinase (5, 6) and the p-adrenergic receptor kinase (PARK) (7, 8),enzymes that have the unique ability to specifically recogof the receptor. Rhonize and phosphorylate the activated form dopsin kinaseand PARK a r e members of a growing familyof G protein-coupled receptor kinases (GRKs) (9-11) that also includes PARK2 (12), IT11 (13), GRK5 (141, and GRK6 (151, as well as two Drosophila kinases, GPRK-1 and GPRK-2 (16). Since G protein-coupled receptors are localized at the plasma membrane, mechanisms for membrane targeting are likely an inherent property of t h e GRKs. For rhodopsin kinase, farnesylation has been shown to be important for its light-dependent translocation to the disc membrane(17, 18). In contrast, association with G protein Py subunits appears to play a role i n membrane targeting and activation of PARK and PARK2 (1923). Since GRKS is neither activatedby G protein P y subunits we have investigated other nor isoprenylated(141, in this study potential mechanisms for regulating the cellular localization and activity of GRK5.

G protein-coupled receptor kinases (GRKs) play an important role in mediating agonist-specific desensitization of numerous G protein-coupled receptors.GRK5, a recently identifiedmember of the GRK family, undergoes a rapid phospholipid-stimulated autophosphorylation to a stoichiometry of -2 mol of phosphate/mol of EXPERIMENTALPROCEDURES GRK5.The ability of phospholipids to stimulate autoMaterials-Restriction endonucleases and other molecular biology phosphorylation is largely blocked by a glutathione S- reagents were purchased from New England Biolabs and Boehringer transferase fusion protein containing the last 102 amino Mannheim. Sequenase was from U. S. Biochemicals, and VentDNA acids of GRKB (amino acids 489-5901, suggesting that polymerase was from New England Biolabs. Isoproterenol,bovine heart this is a primary region involved in GRK5/phospholipid phosphatidylcholine, bovine brain phosphatidylethanolamine, bovine interaction. Phosphoaminoacid determination andmu- liver phosphatidylinositol,bovine brain phosphatidylserine, diacylglycerol, palmitic acid, and myristic acid were from Sigma. All other matetagenesis studies demonstratethatautophosphorylation ofGRK5 occurs primarily at residues Ser-484 and rials were from sources previously described (14, 24). Mutagenesis-TheS484A and T485A double mutation was introThr-485. Expression and characterization of a mutant duced into the full-length clonepGRK5 (14) by PCR-mediated, site(S484A and directed mutagenesis. The sense primer was 5”TGCCTGCCAGGTGRK5 that does notautophosphorylate T485A) reveals that the mutant has a -15-20-fold re- TCGGGC-3’, while the antisense primer 5”ATTGACGCCCduced abilityto phosphorylate the &-adrenergic recep- TTCACAGCTGCGAACTGCTC-3’ encoded the S484A and T485A mutor and rhodopsin comparedto wild type GRK5. These tations (underlined). The PCR reaction contained 10 mM KCI, 10 mM results suggest that phospholipid-stimulated autophos- (NH,),SO,, 20 m Tris-HC1, pH 8.8, 2 mM MgSO,, 0.1% Triton X-100, 400 p~ dNTPs, 50 ng of pGRK5 DNA,1p~ sense and antisense primers, phorylation may represent a novel mechanism memfor and 2 units ofVentDNA polymerase. The -850-bp PCR product was brane association and regulationof GRK5 activity. G protein’-coupled receptors transducea wide varietyof signals ranging from hormones, neurotransmittersand chemoattractants to sensory stimuli such as light, odor, and taste (1,2). The molecular mechanismsof signal transductionby G proteincoupled receptors have been most extensively studied for catecholamine stimulated CAMP production, mediated by the &adrenergic receptor (p#) (31, and phototransduction, mediated by the visual “light” receptor rhodopsin (4). In both systems, the ability of the receptor to respond to its signal is a phenomrapidly diminished upon continuous stimulation, enon termed desensitization. Desensitizationof rhodopsin and the P# is mediated in part by the specific enzymes rhodopsin *This research was supported in part by National Institutes of Health Grant GM44944 (to J. L. B.) and by a predoctoral fellowship from the American Heart Association, Southeastern Pennsylvania Affiliate (to P. K.). The costsof publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Towhom correspondence should be addressed: Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107. Tel.:215-955-4607; Fax: 215-923-1098. The abbreviations used are: G protein, guanine nucleotide-binding protein; &Ut, &-adrenergic receptor; PARK, P-adrenergic receptor kinase; GRK, G protein-coupled receptor kinase; GST, glutathione Stransferase; PCR, polymerase chain reaction; bp, base pair(s).

digested with BsaHI and BgZII, and the resulting -550-bp fragment was purified on a 0.8% low melting agarose gel and used to replace a similar fragment in the clone pGRK5 (14). The PCR-derived portionof the construct was then sequenced in its entirety to confirm the presence of the S484A and T485A mutations. An -880-bp XhoIIKpnI fragment from the mutant pGRK5 clone was then excised, purified, and used to replace a similar fragment in the baculovirusexpression construct pBacPAK-GRK5 (14). ReceptorPhosphorylation-Mutant GRK5 wasoverexpressed and purified from Sf9 cells as previously described forwild type GRK5 (14, 24). Urea-treated rod outer segments were prepared from bovine retinas as previously described (24). The hamster &AR was expressed in Sf9cells, purified by affinity chromatography on an alprenolol-Sepharose column, and then reconstituted into soybean phosphatidylcholine vesicles as described (24, 25). Wild type and mutant GRK5 were routinely assayed using 2-5 p~ rhodopsin, 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 5 mM MgCl,, and 0.1 nm [y-32PlATP(1-1.5 cpdfmol) in a total reaction volume of 20 pl. Rhodopsin phosphorylation reactions were incubated at 30 “C for 1-60 min in the presence of fluorescent room light. The P&R phosphorylation reactions contained 50 nM reconstituted &4R, 100 PM (-)-isoproterenol, 20 nm Tris-HC1,pH7.5, 2 mM EDTA, 5 mM MgCl,, and 0.1mM [y-32PlATP (1-1.5cpdfmol), and were incubated at 30 “C for 5-60 min. Reactions were stoppedby the addition of 10 p1 of SDS sample buffer, and the samples were then electrophoresed on a 10% SDS-polyacrylamidegel (26). The gels were dried and autoradiographed, and the receptor bands were cut and counted. GRK5 Autophosphorylation-Autophosphorylation reactions contained 8-50 ng of GRK5, 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 5 mM (1-2 cpdfmol) in atotal reaction volume MgCI,, and 0.1 mM [Y-~~PIATP of 20 pl. The autophosphorylationreactions were incubated at 30 “C for 3-60 min and then stopped by the addition of 10 pl of SDS sample

10209

Autophosphorylation of GRK5

10210

buffer. The samples were electrophoresed on a 10%SDS-polyacrylamide gel and then analyzed as described above. In experiments involving the activation of GRK5 autophosphorylation, the reactions also contained 20 pg of crude soybean phosphatidylcholine sonicated and reconstituted into liposome vesicles (25). In experiments using purified phospholipids, the phospholipids werelyophilized, resuspended in 20 mM Tris-HC1, pH 7.5, and 2nm EDTA, sonicated, and used a t various concentrations. The chick heart phospholipids were isolated as described earlier (27) and used at a final concentration of 1 pg/ml in the autophosphorylation assay. In experiments involving the fusion protein, a glutathione Stransferase-GRK5 fusion protein(GST-GRK5)containing thecarboxylterminal 102 amino acids of GRK5 (amino acids 489-590) was constructed by inserting a -416-bp BsaHI-SmaI fragment (blunted with Klenow) from pGRK5 into the bacterial expressionvector pGEX-ST a t the SmaI site. The orientationof the construct waschecked by restriction digestion and confirmed by DNA sequencing. The GST-GRK5 fusion protein was expressed inEscherichia coli and purified on a glutathione-Sepharose column using standard procedures (28).

0.5 nn

RESULTS AND DISCUSSION

GRK5 has previously been expressed and purified from Sf9 100 cells and shown to have properties distinct from those of PARK and rhodopsin kinase (24). Further analysis of GRK5 reveals ya 80 that it undergoes a rapid intramolecular autophosphorylation 0 Z 60 reaching a stoichiometry of -1.5 mol of phosphate/mol of kinase after -15 min (Fig. L4). Among the GRKs identified to 40 date, only rhodopsin kinase haspreviously been demonstrated to undergo extensive autophosphorylation (29). The character20 istics of GRK5 autophosphorylation appear similar to thoseof 0 receptor phosphorylation by GRK5, since both have a similar - Liposome + Liposome K,,, for ATP (21 and24 J ~ Mrespectively) , and both are inhibited FIG.1. A, autophosphorylation of GRKB. 40 ng of GRK5 was autoby NaCl (IC5,, values of -70 and -58 mM, respectively) (Ref. 24 and data not shown). However, heparin, a potent inhibitor of phosphorylated in buffer containing 20 nm Tris-HC1, pH 7.5, 2 m~ EDTA, 5 nm MgCl,, and 0.1 nm [y-32PlATP(1 cpdfmol) in a total GRK5-mediated receptor phosphorylation (IC5,, of -1 nM) (24), reaction volume of 20 pl in the presence( 0 )or absence (0) of 20 pg of does not inhibit autophosphorylation even a t a concentration of soybean phosphatidylcholine liposomes. The reactions were incubated 10 J ~ M .Surprisingly, when additional compounds were tested a t 30 "C and stopped at theindicated times by the addition of 10 pl of for their effect on GRK5 autophosphorylation, it was found that SDS sample buffer, followed by electrophoresis on a 10% SDS-polyacrylamide gel and autoradiography. 32Pincorporation in GRK5 was decrude soybean phosphatidylcholine liposomes activated the au- termined by excising and counting theGRK5 bands. The values inditophosphorylation. This resulted inan increase inboth the rate catedaretheaverage of fourindependentexperiments. B , direct and maximal stoichiometry of GRK5 autophosphorylation to binding of GRK5 to liposomes. 40 ng of GRK5 was autophosphorylated for 15 min at 30 "C and then incubated in the presence or absence of 20 -2.5 mol of phosphate/mol of kinase (Fig. lA). pg of soybean phosphatidylcholineliposomes. The samples were centriIn order to assess whether this stimulation of autophospho- fuged a t 100,000 x g for 30 min, and the supernatant and pellet fracrylation might resultfrom the direct bindingof GRK5 to phos- tions were resuspended in SDS sample buffer and processed as depholipid vesicles, GRKS was autophosphorylated with scribed above. Thevaluesindicatedarethemeans ? S.D. of two of liposomes, only -70% of the [Y-~~PIATP, incubated in thepresence or absence of liposomes, independent experiments. In the absence pellet fractions, suggesting and thenpelleted by centrifugation. The resulting supernatant GRK5 was recovered in the supernatant and that some of the kinase was lost, possibly due to association with the and pellet fractions were then analyzed for the presence of surface of the polycarbonate tubes used in the centrifugation. GRKS by SDS-polyacrylamide gel electrophoresis and autoradiography. These studies show that -65% of the GRK5 is pel- acter of the molecule appeared to play the primary role in leted in the presence of liposomes, while minimal amounts of promoting GRK5 autophosphorylation. These studies suggest GRK5 are pelleted in the absence of liposomes (Fig. lB). This that hydrophobic interaction between GRK5 and phospholipid demonstrates that GRKS can directly bind to phospholipids membranes may playan importantrole in promoting autophosand suggeststhat this interaction mightprovide a mechanism phorylation of GRK5. In an attempt to identify the domain in GRKB involved in for promoting GRK5 association with membranes. We next determined the effect of various purified phospho- phospholipid interaction, we generated a construct in which a lipids on GRKS autophosphorylation. Since phospholipids ap- cDNA encoding the COOH-terminal 102 amino acids of GRK5 pear to protectGRKS from salt inhibition (data not shown), this (amino acids 489-590) was ligated to the glutathione S-transstudywas performed a t physiological salt concentrations. ferase gene(GST). This region of GRK5 was chosen becausethe Crude soybean phosphatidylcholine, chick heart phospholipids, COOH-terminal domains of both rhodopsin kinase (via farneand purifiedphosphatidylcholine andphosphatidylethanolsylation) (18)and PARK (via interaction with subunits) (20, be involved in membranelocalization. The resultamine all promoted a -34-fold activation of GRK5 autophos- 30) appear to phorylation, albeit at different optimalconcentrations (Fig. ing GST-GRK5 fusion protein was able to significantly inhibit 2 A ) . Similar resultswere obtained with purified phosphatidyl- phospholipid-mediated activation of GRK5 autophosphorylainositol and phosphatidylserine (data not shown). Since these tion (Fig. 2B). This effect appeared to be specific, since GST results suggested a lack of specificity toward the head group of alone had no effect on GRKS autophosphorylation.When a Fig. 3)was the phospholipids, we tested several other hydrophobic mol- mutant GRK5 that does not autophosphorylate (see ecules. Diacylglycerol was also found to activate GRK5 auto- tested, it was found to be slightly more potent than the GSTas myristic GRKS fusion protein a t inhibiting thephospholipid-stimulated phosphorylation, as did long chain fatty acids such and palmitic acid (Fig.2 A ) . In each case, the hydrophobic char- autophosphorylation (Fig.2B). While theseresults demon-

Autophosphorylation of GRK5 A

10211 Rhodopsin kinase GRKS

B

1

0

484 480

VGAFSTVKGV IEQFSTVKGV

2

6 7

kDa

-rs

kDa

-30

kDa

FIG.3.A, sites of CRK5 autophosphorylation. The University of Wisconsin Genetics Computer Group software package was used to a l i w the carboxyl-terminal sequence flanking the two major autophosphtr rylation sites in rhodopsin kinase (indicatedin h o l d ) with the carhoxylterminal domain of GRK5. B . autophosphorylation mutant of CRKS. Ser-484 and Thr-485 in the human clone pCRK5 ( 14) were mutated to alanines by PCR-mediated site-directed mutagenesis. and the resulting mutant GRK5 was expressed and purified from SI9 cells as described under "Experimental Procedures."30 ng of wild type CRK5 (Innc, 1 1 or mutant CRK5 (lane2 ) were incubated with 20 mM Tns-HCI. pH 7.5,2 mM EDTA, 5 mM MgC12, and 0.1 mM Iy-'"I'IATI'( 1 cpdfmtrl I at 30 'C for 60 min. The reactions were stopped by the addition of SDS sample buffer followed by gel electrophoresisandautoradiopaphy.Similar studies have demonstrated that the mutant CRK5also does not autophosphorylate in the presence of liposomes.

minor site is not conserved. Since phosphoamino acid analysis of autophosphorylated GRK5 revealed both phosphoserine and phosphothreonine (data not shown), we generated a double mutation converting both Ser-484 and Thr-485 to alanines by site-directed mutagenesis. This construct was then subcloned into a baculovirus expression vector, and the mutant kinase was expressed and purified from Sf9 cells. The mutant GRK5 did not autophosphorylate either in the presence or absence of 0 1 10 100 liposomes, thereby confirming Ser-484 and Thr-485 as the priMolar ratio mary sites of autophosphorylation in GRKB (Fig. 3 R ) . FIG.2. A, activation of GRKB autophosphorylation by various phosMultiple protein kinases have been shown to alter their catapholipids. 50 ng of GRKS was autophosphorylated a s described in the lytic activity after autophosphorylation (29, 32-35). For exFig. 1 legend, exceptthat the reactions also contained 150 mM NaCl and of protein kinaseC were inthe absence or presence of 20 pgof soybean phosphatidylcholine ample, intramolecular autophosphorylation liposomes, 20 ng of chick heart phospholipids, 0.2 pg of phosphatidyl- activates the enzyme,a mechanism that appearsto serve as a choline, 0.2pg of phosphatidylethanolamine, 20 ngof sn-l,2-diacylglyc- positive feedback typeof regulation (35).In contrast, rhodopsin ern1 (C2O:O). 20 ng of sn-1.2-diacylglyceroi (C28:O),20 ng of myristic kinase autophosphorylation has been proposed to play a role in acid, or 20 ng of palmitic acid. Samples were incubated a t 30 "C for 5 regulating its binding to rhodopsin, since autophosphorylated min and then processed a s described earlier. One-fold represents the stoichiometry of GRK5 autophosphorylation in the absenceof any acti- rhodopsin kinase appears to bind tightly to rhodopsin but has vator (0.12mol of phosphate/mol of GRK5). The values indicated are the a lower affinity for phosphorylated rhodopsin (29). means * S.D. of three independent experiments. B , inhibition of l i p To determine the roleof GRK5 autophosphorylation, mutant some-activated autophosphorylation by the carboxyl-terminal domain and wild type GRK5 were assessed for their ability to phosphoof GRK5. 60 ng of GRK5 was autophosphorylated as described above rylate various exogenous substrates. When a non-receptor suh(including 150 mM NaCI) in the presence or absence of 20 ng of chick heart phospholipids. As indicated, incubations contained a 1-100-fold strate such as casein was used, the mutant GRK5 was found to molar ratio of either the CST-GRK5 fusion protein (A),mutant GRK5 be -2-fold less active than wild type GRKB (data not shown). (0). or CST (A). The samples were incubated a t 30 "C for 15 min and processed by SDS-gel electrophoresis. One-fold represents the stoichi- This demonstrates that the mutantGRK5 retains much of its is propometry of CRK5 autophosphorylation in the absence of any activator catalytic activity and suggests that the mutant kinase (0.22 mol of phosphate/mol of GRK5). TheGST-GRK5 fusion protein or erly folded. In contrast, the mutant GRK5 was dramatically GST did not inhibit GRK5 autophosphorylation in the absence of lipo- reducedinitsability to phosphorylatereceptorsubstrates. somes. The values indicated are the means S.D. of three independent While GRKB rapidly phosphorylates the &AR in a n agonistexperiments. dependent mannerto a stoichiometry of -2.5 mol of phosphate/ mol of &AR, the mutant GRK5 was -20-fold less active, restrate that the COOH-terminal -100-amino acid domain of sulting in a stoichiometry of only 0.12 mol of phosphatdmol of GRK5 can directly interact with phospholipids, we cannot rule &AR after a 60-min incubation (Fig. 4A ). However, similar to out the possibility that additional domains of GRK5 may also wild type GRK5. the mutant GRK5 still specifically phosphorylatedonlytheactivatedform of thereceptor(data not be involved in phospholipid interaction. shown). Analogous results were obtained when rhodopsin was Previousstudieshaveidentifiedtwomajorautophosphorylation sites in the carboxyl-terminal domain of rhodopsin used as the substrate (Fig. 4R). While GRK5 phosphorylates rhodopsin to a stoichiometry of -1 mol of phosphatehol of kinase (Ser-487 and Thr-488) with an additional minor site near the amino terminus (Ser-21) (31). A comparison of the rhodopsin, the mutant GRK5 was - 17-fold less active. resultGRKB and rhodopsin kinase sequences reveals that the two ing in a stoichiometry of only 0.06 mol of phosphatehol of major autophosphorylation sites in rhodopsin kinase are con- rhodopsin after 60 min. Kinetic analysisof rhodopsin phosphoGRK5 had an -2.R-fold rylation demonstrated that the mutant served in GRKB (Ser-484 and Thr-485) (Fig. 3 A ) , while the

10212

Autophosphory 'lation of GRKS tential mechanism for the in vivo targeting of GRKs to their receptor substrates. It will be interesting to assess whether a similar mechanism is involved in regulating other closely related members of the G protein-coupled receptor kinase family such as IT11 and GRK6. While there are many structural and functional similarities among the GRKs, our findings suggest that distinct mechanisms may serve to regulate the activity, substrate specificity, and cellular localization of various members of the GRK family.

-

2.5

u 2.0 -

2" -

.

1.5

-

E

1.0

-

._ a -

t/

0.5

.-.--.'

"

'

0.0

0

1030 20

40

Time ( min

'

7

50

60

Acknowledgments-We thank Dr. James J. Onorato for kindly providing the purified hamster &-adrenergic receptors, Dr. Marlene M. Hosey for providing the purified chick heart phospholipids, and Drs. Thomas Frielle, James Keen, and Susan Rittenhouse for valuable discussions.

)

,

1.0

i1

B

I

REFERENCES

1. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991)Annu. Reu. Biochem. 60, 653-688 2. ODowd, B. F., Lefkowitz, R. J., and Caron, M. G. (1989) Annu. Reu. Neurosci. o/o"o 0.8 2, 67-83 0 ' / 3. Gomez, J., and Benovic, J. L. (1992) in Molecular Biology of Receptors and 0 Dansporters: Receptors (Friedlander, M., and Muekler, M., eds) pp. 1-34, E 0.6 Academic Press, San Diego 4. Hargrave, P. A., and McDowell, J. H. (1992) in Molecular Biology ofReceptors A and Dansporters: Receptors (Friedlander, M., and Muekler, M., eds) pp. a 49-98, Academic Press, San Diego - 0.4 5. Kuhn, H., and Dreyer, W. J . (1972) FEBS Lett. 20, 1 4 6. Bownds, D., Dawes, J., Miller, J., and Stahlman, M. (1972) Nature 237, 125127 o.2 7. Benovic, J . L., Strasser, R. H., Caron, M. G . , and Lefkowitz, R. J. (1986) Proc. e -.Natl. Acad. Sci. U. S. A. 83, 2797-2801 0.0' ' ' ' ' 8. Lohse, M. J., Benovic, J. L., Caron. M. G . , and Lefkowitz, R. J. (1990) J . Biol. 0 10 20 30 40 50 60 Chem. 265, 3202-3209 Time ( min ) 9. Palczewski, K., and Benovic, J . L. (1991) '12ends Biochem. Sci. 16, 387-391 J. L., DeBlasi, A,, Stone, W. C., Caron, M. G., and Lefkowitz, R. J . FIG.4. Comparison of the activities of wild type and mutant 10. Benovic, (1989) Science 246, 235-240 GRKB. A, phosphorylation of the &-adrenergic receptor. 50 nM P 2 A R 11. Lorenz, W., Inglese, J., Palczewski, K., Onorato, J. J.,Caron, M. G., and was phosphorylated with25 nM wild type (0) or mutant ( 0 )GRK5 in the Lefkowitz, R. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 87154719 presence of 100 PM (-)-isoproterenol. B , rhodopsin phosphorylation. 3 12. Benovic, J. L., Onorato, J. J., Arriza, J . L., Stone, W. C., Lohse, M., Jenkins, N. PM rhodopsin was phosphorylated with 50 nM wild type (0) or mutant A,, Gilbert, D. J.,Copeland, N. G., Caron, M. G., and Lefkowitz, R. J . (1991) J. Biol. Chem. 266, 14939-14946 ( 0 )GRK5 in fluorescentroom light. The values indicated are the aver13. Ambrose, C., James, M., Barnes, G . , Lin, C., Bates,G., Altherr, M., Duyao, M., age of two independent experiments. Groot, N., Church, D., Wasmuth, J . J., Lehrach, H., Housman, D., Buckler, A,, Gusella, J. F., and MacDonald,M. E.(1992)Hum. Mol. Genet. 1,697-703 higher K,,, and -5.3-fold lower V,,, compared to wild type 14. Kunapuli, P., and Benovic, J. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5588-5592 GRK5. These studies suggest that autophosphorylation may 15. Benovic, J. L., and Gomez, J . (1993) J. Biol. Chem. 268, 19521-19527 play a critical role in promoting the phosphorylation of G pro- 16. Cassill, J. A., Whitney, M., Joazeire, A. P., Becker, A., and Zuker, C. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11067-11070 tein-coupled receptors by GRK5. 17. Inglese, J., Glickman, J. F., Lorenz, W., Caron, M. G . , and Lefkowitz, R. J. Autophosphorylation is a feature common to a wide variety (1992) J. Biol. Chem. 267, 1422-1425 of serinekhreonine and tyrosine protein kinases. In this report 18. Inglese, J., Koch, W. J., Caron, M. G., and Lefkowitz, R. J. (1992) Nature 359, 147-150 we have demonstrated that GRK5 also undergoes rapid in19. Haga, T., and Haga, K. (1992) J. Biol. Chem. 267,2222-2227 tramolecular autophosphorylation to a stoichiometry of -2 mol 20. Pitcher, J. A,, Inglese, J., Higgins, J . B., Arriza, J . L., Casey, P. J., Kim, C. M., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J . (1992) of phosphate/mol of kinase. Although two primary sites of auScience 257, 1264-1267 tophosphorylation were identified by phosphoamino acid anal- 21. Kim, C. M., Dion, S. B., and Benovic, J. L. (19931J. Biol. Chem. 268, 15412ysis and confirmed by mutagenesis, the rate of GRK5 autophos15418 22. Kim, C. M., Dion, S. B., Onorato, J . J., and Benovic, J . L. (1993) Receptor 3, phorylation suggests that the first site isautophosphorylated 39-55 rapidly, followed by slower phosphorylation of an additional 23. Boekhoff, I., Inglese, J., Schleicher, S., Koch, W. J., Lefkowitz, R. J., and Breer, H. (1994) J . Biol. Chem. 269, 3 7 4 0 site. Since previous studies have shown that GRK5 does not P., Onorato, J. J., Hosey, M. M., and Benovic, J . L. (1994) J. Biol. readily phosphorylate residues in an acidic environment (24), it 24. Kunapuli, Chem. 269, 1099-1105 is conceivable that the presence of a phosphate group on the 25. Cerione, R. A,, Strulovici,B., Benovic, J. L., Lefkowitz, R. J., and Caron, M. G . (1983) Nature 306, 562-566 primary site may hinder the transferof phosphate to an adja26. Laemmli, U. K. (1970) Nature 227, 680-685 cent site. Previous studies on protein kinase C have suggested 27. Kwatra, M. M., Benovic, J. L., Caron, M. G., Lefkowitz, R. J., and Hosey, M. M. that the proximity of the autophosphorylated residue to the (1989) Biochemistry 28, 45434547 ATP binding pocket in the catalytic domain appears to be the 28. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.167, 3 1 4 0 29. Buczvlko. J.. Gutmann. C.. and Palczewski, K. (1991) Proc. Natl. Acad. Sci. primary criterion for autophosphorylation (36). A similar sceU.-S. A. 68, 2568-2572 nario would place Ser-484 and Thr-485 in close proximity to the 30. Koch, W. J., Inglese, J., Stone, W. C., and Lefkowitz, R. J. (1993) 268, 8 2 5 6 8260 catalytic site in GRK5. Moreover, the decreased ability of mu31. Palczewski, K., Buczylko, J., Van Hooser, P., Carr, S. A,, Huddleston,M. J.,and tant GRK5 to phosphorylate receptor substrates suggests that Crabh, J . W. (1992) J . Biol. Chem. 267, 18991-18998 the autophosphorylation domain may also play an important 32. Wang, J. H., Stull, J, T., Huang, T. S., and Krebs, E.G. (1976) J. Biol. Chem. 251,45214527 role in receptor recognition. 33. Rangel-Aldao, R., and Rosen, D. M. (1976) J. Biol. Chern. 251, 3375-3380 These studies thus demonstratea novel mechanism of regu- 34. Kuret, J., and Schulman, H. (1985) J . Biol. Chem. 260, 6427-6433 lation of G protein-coupled receptor kinase activity. Phospho- 35. Mochly-Rosen, D., and Koshland, D. E.,Jr. (1987) J. Biol. Chem. 262, 22912297 lipid-stimulated autophosphorylation of GRK5 may provide a 36. Flint, A. J., Paladini, R. D., andKoshland, D. E., Jr. (19901 Science 249, 408411 mechanism for both activation of the kinase, as well as a po-

-

.z 3

E E

'

P

1

.