Vol. 183, No. 2, 1992

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 16, 1992

Pages 468-473

IN VZVO FARNESYLATION

OF RAT RHODOPSIN

KINASE

JanmeetS. Anant and Bernard K.-K. Fung JulesStein Eye Institute University of California, Los Angeles, CA 90024 Received

January

10,

1992

We have shown by intravitreal injection of [31-Ilmevalonolactonethat a 65 kDa protein in rat photoreceptorsis posttranslationally modified by famesylation. We further identified this 65 kDa prenylated protein asrhodopsinkinasebasedon its affinity for photolyzed rhodopsinand its ability to autophosphorylatein the presenceof [Y-~~P]ATP. The famesylation of rhodopsin kinasemay be important for correctly targeting this enzyme to the photoreceptorouter segments,allowing it to phosphorylatephotolyzed rhodopsinefficiently. o 1991Academic press,M.

Light activation of rhodopsin in vertebrate retinal photoreceptors triggers a cyclic GhIP cascadethat ultimately leads to an amplified electrical response(1). However, in order for a photoreceptorto becomeresponsiveagainwithin secondsafter the light stimulationis terminated,it must also possessa cellular mechanismto deactivate the photolyzed rhodopsin. This processis known to be mediated by the phosphorylation at multiple serine and threonine sites near its carboxyl terminal region (2, 3) and the subsequentbinding of arrestin to the phosphorylatedform of opsin (4). The concertedeffects of phosphorylationand arrestinbinding block further activation of the cyclic GMP cascade,thusallowing the photoreceptorto recovery to the dark-adaptedstate. The phosphorylation of photolyzed rhodopsinis catalyzed by rhodopsinkinase, a memberof the G protein-coupled receptor kinasefamily. It is a 65 kDa protein found mainly in the rod outer segments(ROS) and loosely associatedwith the disk membranes(5). Recently, the deduced amino acid sequenceof bovine rod rhodopsin kinase hasbeen determined (6). A salient feature emerging from the sequenceanalysis is the presenceof a cysteine residue located at the fourth position from the carboxyl terminus. This CXXX (C=cysteine and X=any amino acid) motif is known to be a signal sequencefor a complex seriesof posttranslationalmodifications (7, 8). In this process,a famesyl or geranylgeranyl group is covalently attached to the sulfhydryl group of the cysteine residuethrough a thioether linkage. The last three amino acid residuesarc removed, and the newly exposed cx-carboxyl group on the terminal cysteine is methyl esterified. In this report, we provide evidence showing that rat rhodopsin kinase is prenylated in vivo. We further identify the prenyl moiety as a famesyl group attached to a cysteine residue. Famesylation of rhodopsin kinase may play an important role in the proper functioning of this enzyme during phototransduction. 0006-291X192 Copyright All rights

$1.50 0 1992 bv Academic Press. of reproduction in any form

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468

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EXPERIMENTAL

AND BIOPHYSICAL

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PROCEDURES

In Viva Prenylation of ROS Proteins. Sprague-Dawley rats were anesthetized with ether and injected intravitreally with 2 ~1 per eye of a saline solution containing 2 pg of mevinolin (9) and 20 PCi of [3H]mevalonolactone (New England Nuclear, specific activity of 35 Ci/mmol). The rats were maintained in darknessfor 20 h and sacrificed with CO2. The retinas were removed under dim red light, suspendedin an ice-cold 12% sucrosesolution (1 retina/ml of solution), and shaken vigorously for 2 min. The detached ROS were fractionated by centrifugation on a discontinuoussucrosedensity gradient, collected from the 28140%sucroseinterface, and analyzed by SDS-PAGE (10). The radioactivity associatedwith the protein bandswasquantitated either by excision of the appropriate gel regions followed by digestion in 20% hydrogen peroxide at 65°C overnight and liquid scintillation counting, or by autoradiography using Kodak XAR-5 film following equilibration of the gel with fluorographic reagentAmplify (Amersham). Extraction of Rhodopsin Kinase. Rhodopsin kinase was routinely solubilized by incubating the dark-adapted ROS membranes(6 retina-equivalents) in 100 ~1 of hypotonic buffer (2 mM sodium phosphate,pH 7.6, 1 mM MgC12, 2mM DlT, 0.1 PM Okadaic acid) for 15 min at O”C, followed by the addition of 100 ~1of hypotonic buffer containing 140 mM sodiumphosphateand centrifugation in a Beckman Airfuge at 20 psi for 5 min. The supematantwas removed and the extraction was repeated. The two extracts were then combined (400 ~1) and centrifuged in a Beckman Airfuge at 20 psi for 5 min to remove any remaining ROS membranes.This preparation was usedin the autophosphorylationreaction describedin the next section. Binding of Rhodopsin Kinase to ROS Membranes. The binding of rhodopsin kinase to dark-adaptedor photolyzed ROS membraneswas measuredby a modified procedureof Kiihn (5). Briefly, ROS (6 retina equivalents)were incubatedin 600 @ of hypotonic buffer for 15 min at O”C, freeze-thawed 3 times to lyse the ROS, and divided into equal aliquots. One aliquot was kept in darknessand the other was photolyzed for 5 min. Thesetwo sampleswere then centrifuged on a Beckman Airfuge at 20 psi for 5 min to pellet the membranes.The radioactivity associatedwith rhodopsin kinase in the supematantand the pellet wasdetermined after separationof the proteins by SDS-PAGE. Autophosphorylation of Rhodopsin Kinase. Autophosphorylation of rhodopsin kinase was initiated by the addition of 10 ~1 of 0.1 mM [r-32P]ATP (New England Nuclear, specific activity of 3-4 Wmmol ATP) to 90 ~1of the protein extract. After a 30-min incubationat 22”C, the reaction was terminated by adding 34 ~1 of SDS-samplebuffer (10). The radiolabeled proteins were separatedby SDS-PAGE and detected by autoradiography or scintillation counting. As a control, the membranepellet was also resuspendedin 400 ~1 of hypotonic buffer containing 70 mM sodiumphosphateandphosphorylatedasdescribedabove. Confirmation of the Identity of Rhodopsin Kinase by Reverse-phase HPLC. ROS proteins were radiolabeled by prenylation and phosphorylation and separated on a SDSpolyacrylamide slab gel (10). Immediately following electrophoresis,a strip was removed and stained to locate the position of the radiolabeled rhodopsin kinase band. Gel pieces containing rhodopsinkinasewere excisedfrom the unstainedgel and the polypeptide waseluted by incubation for 8 h with extraction buffer containing 0.1 M Tris-acetate, pH 6.0, 0.2% SDS, 1% Triton X100. After removal of the gel piecesby filtration, SDS in the extraction buffer was precipitated by the addition of 0.1 ml of 2 M KCl/ml of filtrate. Approximately 80% of the total radiolabeled rhodopsin kinase was recovered at this stage. This samplewas then applied onto a Aquapore Octyl RP-300 reverse-phase HPLC column (Brownlee Labs) equilibrated with 0.1% (w/v) trifluoroacetic acid in water and eluted with a linear O-80%acetonitrile gradient at a flow rate of 1 ml/min over 40 min. One-minute fractions were collected and assayedfor radioactivity. HPLC Analysis of the Prenyl Group. The prenyl group of rhodopsin kinase was identified by the procedure of Anant et al.( 11). Briefly, a sampleof the [3H]prenylated rhodopsin kinase

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(2000 cpm) was purified by preparative SDS-PAGE as described in the previous section. To convert the polypeptide to its amino acid constituents, the purified rhodopsin kinase was digested with 2.5 mg/ml Pronase (Srrepromyces griseus, Calbiochem) for 5 h at 37°C. These digestion conditions have been shown to hydrolyze carboxyl methyl esters (12). The final digests were analyzed immediately by reverse-phaseHPLC asdescribedpreviously (13, 14). Fractions (1 ml) were collected and assayedfor radioactivity. The identity of the prenyl group was confirmed by comparing the radioactivity profile with the elution profiles of authentic famesyl cysteine and geranylgeranyl cysteine.

RESULTS We have recently developed an intravitreal injection procedure to metabolically label the prenyl group of retinal proteins and showed that the a and p subunits of cyclic GMP phosphodiesteraseare modified by famesylation and geranylgeranylation, respectively (11). During the course of our investigation, we observed that another polypeptide with an apparent molecular weight of 65 kDa in the ROS preparation was alsometabolically labeledby intravitreal injection of [3H]mevalonolactone. The amount of radioactivity associatedwith this 65 kDa polypeptide was approximately 20% of that in the phosphodiesterase (Fig. 1, lane l), suggesting that it is only a minor protein componentof the ROS. Since bovine rhodopsin kinase has the sameapparentmolecular weight (5) and contains a carboxyl-terminal CVLS sequence(6) postulated to be the signal for the post-translational modification by protein prenylation (7,8), it appearslikely that the 65 kDa prenylated polypeptide we have observed is rat rhodopsin kinase. To test this hypothesis, we measuredthe solubility of this retinal protein under various conditions. We found that it can be eluted with moderateor low

M,x -

IO3 92

-

66

-

45

Figure 1. Prenylation and autophosphorylation of ROS proteins. [3H]prenylated ROS proteins (lane 1) were separated into soluble (lane 2) and membrane fractions as described in “Experimental Procedures.” The soluble fraction (lane 3), the soluble fraction reconstituted with urea-washed ROS membranes (lane 4), and the membrane fraction (lane 5) were then incubated with [Y-32P]ATP at 22°C for 30 min. The radiolabeled proteins were separated by SDS-PAGE and analysed by autoradiography. To mask the [%IJradioactivity in lane 3-5, the gel was covered with a thin sheet of plastic during explosure to the film. The arrowhead indicates the position of radiolabefed rhodopsin kinase (RK). 470

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600

300

3 +

400

200

a ';;

6a = ITz

200

100

3 6 6 A

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E

02 O loElution2oTime3o(min)4o 5o

0

10

20

30

40

50

ElutionTime (mm)

Figure 2. Confirmation by reverse-phase HPLC analysis that rhodopsin kinase is both prenylated and phosphorylated.The radiolabeled65 kDa polypeptidewaselutedfrom the SDS-polyacrylamidegel andchromatographed asdescribedin “ExperimentalProcedures.” Fractions(1 ml) werecollectedandassayed for radioactivity. The arrowheaddenotestheelution time of famesylated y subunitof transducin chromatographed underidenticalconditions. Figure 3. Chromatographic identification of the prenyl group attached to rhodopsin kinase. The [3H]prenylated65 kDa ROSpolypeptidewaspurifiedby SDS-PAGE,digestedwith Pronase,andanalyzedby reverse-phase HPLC asdescribedunder“ExperimentalProcedures.” Fractions(1 ml ) werecollectedandassayed for radioactivity. The arrowheads indicatetheelution times of authentic famesylcysteinefFc) andgeranylgeranyl cysteine(GGC)asdetectedby theUV absorbance at 215nm.

ionic strength buffers (Fig. 1, lane 2). Approximately 50% of the total radioactivity associated with the 65 kDa protein in the ROS can be solubilized after the first extraction. However, when the extractions were carried out in the light under identical conditions, only 25% of the total radioactivity wasfound in the supematant.This result indicatesthat the 65 kDa prenylated protein has a higher affinity for the photolyzed ROS membranes,as would be expected if it is rat rhodopsinkinase (5). Another distinctive characteristicof rhodopsinkinaseis its ability to autophosphorylatein the presenceof ATP (15, 16). To further confirm the identity of the 65 kDa polypeptide, we extracted the [3H]prenylated proteins with hypotonic buffer and incubated the extract with [Y-~~P]ATP at 22°C for 30 min, followed by SDS-polyacrylamide gel electrophoresisanalysisof the radiolabeled proteins ((Fig.1, lane 3). As a control, we also incubated the kinase-depletedROS membranes with [y-32P]ATF’ under identical conditions (lane 5). As shown in lane 3 of Fig. 1, over 70% of the radioactivity in the hypotonic protein extract wasassociatedwith the 65 kDa prenylated protein, suggestingthat it is a phosphoprotein. Moreover, there was a marked decreaseof radioactivity in this protein band in membranespartially depletedof rhodopsinkinase (lane 5). The radioactivity associatedwith the 65 kDa protein, aswell asrhodopsinphosphorylation, was recovered upon the reconstitution of the extract with the kinase-free, urea-washedmembranes(lane 4). This result confirms that the extract containsrhodopsinkinaseactivity. To further ascertainthat the prenylated and phosphorylated65 kDa protein were not different polypeptides comigrating in the SDS-polyacrylamide gels, we extracted the dual radiolabeled65 kDa polypeptide from the SDS-polyacrylamide gel and rechromatographedthe protein extract on reverse-phaseHPLC. Fig. 2 shows that both the [32P] and [3H]radiolabels co-eluted as a single 471

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peak of radioactivity at 30 min. In comparison, the [%Ijradiolabel y subunit of transducin molecular weight,

associated with the farnesylated

(17, 18) eluted from the column at 26 min.

membrane-binding

properties,

COMMUNICATIONS

specific localization

Based on its apparent to ROS, and ability to

autophosphorylate, we conclude that the 65 kDa prenylated protein is rat rhodopsin kinase. To determine the structure of the prenyl group, we purified rhodopsin kinase by preparative SDS-polyacrylamide

gel electrophoresis, exhaustively digested the polypeptides to their constituent

ammo acids, and fractionated the digests by reverse-phase HPLC (13, 14). The polypeptides were digested with Pronase under conditions that have been shown to hydrolyze carboxyl methyl esters (12).

Analysis

of the radioactive

materials obtained from digestion of the rhodopsin kinase

polypeptide revealed a single peak of radioactivity which eluted at the same position as the famesyl cysteine standard, indicating that rat rhodopsin kinase is modified by famesylation (Pig. 3).

DISCUSSION Based on the evidence presented here, we have concluded that rat rhodopsin kinase contains a farnesyl group covalently modification

attached to a cysteine residue through a thioether linkage.

is most likely mediated by a protein farnesyltransferase

This

that catalyzes the transfer of

the farnesyl group from famesyl pyrophosphate to rhodopsin kinase. Protein famesyltransferase has recently been isolated from the cytosolic fraction of the brain homogenates (19) and shown to have specificity

towards

short peptides or protein substrates containing the CAAX

sequence at the carboxyl-terminus,

where A represents aliphatic amino acids and X represents

polar residues such as Gln, Met, and Ser (20). rhodopsin kinase is still not known, deduced carboxyl-terminal

consensus

Although

the amino acid sequence of rat

the farnesylation of rhodopsin kinase is consistent with the

CVLS sequence of bovine rhodopsin kinase (6) and the substrate

specificities of the protein farnesyltransferase

purified from rat brain (19,20).

Rhodopsin kinase and P-adrenergic receptor kinase are two members of the receptor kinase family. They share many similarities in structure, function, and biochemical properties. However, unlike rhodopsin kinase, /3-adrenergic receptor kinase does not contain a CXXX

amino acid

sequence at the carboxyl terminus (21) and therefore is unlikely to be modified by prenylation. padrenergic receptor kinase has also been reported to fractionate with the supernatant of high speed centrifugation

of lysed cells (22), indicating that it is a cytosolic protein. In contrast, rhodopsin

kinase is loosely associated with dark-adapted ROS membranes (5) and, in our hands, solubilized only after repeated extractions

with moderate ionic strength buffer.

As has been reported

previously, the binding of rhodopsin kinase to ROS membranes increases after bleaching due to a specific interaction of the enzyme with photolyzed rhodopsin (5). Recent studies have suggested that the addition of the farnesyl group confers a moderate degree of membrane avidity on the modified

protein (23, 24), in agreement with the small difference

in membrane

binding

characteristics between rhodopsin kinase and p-adrenergic receptor kinase. A number of proteins involved in the phototransduction include transducin (17, 18), cyclic GMP phosphodiesterase 472

process of retinal rods, which

(11) and rhodopsin kinase, have now

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been shown to be modified by prenylation. modification?

AND

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What might be the function of this highly specific

We speculate that prenylation may play a role in bringing these proteins to the disk

membranes near the rhodopsin molecules, where they can interact efficiently.

ACKNOWLEDGMENTS We wish to thank Dr. Harvey Yamane for his valuable discussion and Alice Van Dyke for her excellent photographic assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 1.5. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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