Stimulus-dependent Phosphorylation of G-protein-coupled Receptors by Casein Kinase 1alpha *

(Received for publication, March 31, 1997, and in revised form, June 2, 1997)

Andrew B. Tobin Dagger §, Nicholas F. Totty , Alistair E. Sterlin and Stefan R. Nahorski Dagger

From the Dagger  Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom and  The Ludwig Institute for Cancer Research, University College London Medical School, 91 Riding House Street, London W1P 8BT, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of ~40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta -adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha ) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha . 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha , and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.


INTRODUCTION

Intensive research over the last decade have revealed that many GPCR1 subtypes are phosphorylated in response to agonist stimulation (1). These receptors include those coupled to either the adenylate cyclase or phospholipase C (PLC) pathways and suggests that receptor phosphorylation is a common regulatory mechanism employed by all but a few GPCR's (1). For the majority of these receptors the cellular protein kinases involved in agonist-mediated receptor phosphorylation have yet to be determined. However, this is not the case for the extensively studied beta -adrenergic receptor where agonist-dependent phosphorylation and receptor desensitization is mediated by the receptor-specific kinase, beta -adrenergic receptor kinase (beta -ARK) (2, 3).

Studies using purified or partially purified receptor preparations reconstituted in phospholipid vesicles with purified beta -ARK, have demonstrated that beta -ARK is also able to phosphorylate both cyclase-coupled (e.g. m2-muscarinic (4)) and PLC-coupled (e.g. substance P receptor (5)) receptors in an agonist-dependent manner. Furthermore, the use of dominant negative mutants of beta -ARK to inhibit endogenous beta -ARK activity (6) has suggested that beta -ARK is the endogenous kinase responsible for the phosphorylation of recombinant PLC-coupled alpha 1B-adrenergic receptors expressed in COS-7 cells and rat-1 fibroblasts (7), angiotensin II receptors in HEK 293 cells (8), and the cyclase-coupled delta -opioid receptors in HEK 293 cells (9). These studies have indicated that beta -ARK may have a broad receptor substrate specificity that extends beyond beta -adrenergic receptors.

beta -ARK-1 and beta -ARK-22 are members of a protein kinase family called the G-protein-coupled receptor kinase (GRK) family (10). The newly cloned members of the GRK family, namely IT-11 (GRK-4), GRK-5, and GRK-6, phosphorylate rhodopsin in reconstituted systems (11-14). Furthermore, GRK-5 is able to phosphorylate purified alpha 2-and beta 2-adrenergic, and m2-muscarinic receptors (11-13) and GRK-6 to phosphorylate beta 2-adrenergic and m2-muscarinic receptors, in an agonist-dependent manner (14). Therefore, it appears that beta -ARK and other members of the GRK family are able to phosphorylate a number of GPCR subtypes in reconstituted systems lending support to the proposed role of this protein kinase family in phosphorylation of GPCRs.

However, recent evidence suggests that receptor phosphorylation mediated by beta -ARK and the GRKs may be inhibited by the PLC signaling pathway. For example, increased intracellular calcium concentrations and depletion of the phospholipid, phosphoinositide 4,5,-bisphosphate (PIP2), may contribute to a reduction in the activity of GRKs (see "Discussion"). This raises the possibility that agonist-dependent phosphorylation of PLC-coupled receptors may be mediated by an alternative protein kinase pathway from that of beta -ARK and the other GRKs.

Our early studies on the PLC-coupled m3-muscarinic receptor indicated that the rapid serine phosphorylation observed following agonist stimulation (15) was mediated by a receptor kinase that was distinct from beta -ARK (15, 16). We have recently purified a 40-kDa protein kinase from porcine cerebellum that is able to phosphorylate a glutathione S-transferase bacterial fusion protein containing a portion of the third intracellular loop of the m3-muscarinic receptor (17). Furthermore, this 40-kDa protein kinase was able to enhance the agonist-dependent phosphorylation of the muscarinic receptor present in membranes obtained from CHO-m3 cells transfected with the human m3-muscarinic receptor cDNA (17). The molecular weight, chromatographic properties, and protein kinase inhibitor studies demonstrated that the 40-kDa protein kinase was distinct from the second messenger-regulated protein kinases (e.g. protein kinase C) and from beta -ARK and other members of the GRK family (17). These findings indicated that the 40-kDa protein kinase, called muscarinic receptor kinase (MRK), represents a previously unidentified receptor-specific kinase that offers an additional/alternative protein kinase pathway for the phosphorylation of the m3-muscarinic receptor and possibly other GPCRs.

In the present paper we present evidence that MRK is a member of the casein kinase 1 family, namely casein kinase 1alpha (CK1alpha ), and reveal the ability of the recombinant kinase to phosphorylate m3-muscarinic receptors and rhodopsin in an agonist/stimulus-dependent manner.


EXPERIMENTAL PROCEDURES

Cell Culture

CHO (Chinese hamster ovary) cell cultures stably transfected with human m3-muscarinic receptor cDNA (CHO-m3 cells, a kind gift from Dr. N. J. Buckley, Dept. Pharmacology, University College, London, UK) contained ~2100 fmol of receptor/mg of protein. These cells were routinely maintained in alpha -minimal essential medium supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), fungizone (2.5 µg/ml), and fetal calf serum (10% v/v). Spodoptera frugiperda (Sf9) cells were maintained as suspension cultures in SF900 II medium (Life Technologies, Inc.).

Purification of Muscarinic Receptor Kinase (MRK) from Porcine Brain

The procedure used for purification of MRK from porcine cerebellum has been previously described (17).

Preparation of the Bacterial Fusion Protein Ex-m3

Preparation of the bacterial fusion protein, Ex-m3, where amino acids Ser345-Leu463 of the human m3-muscarinic receptor third intracellular loop are fused with glutathione S-transferase has previously been described (15).

Amino Acid Sequencing

The excised Coomassie-stained protein band corresponding to MRK was digested with trypsin. Peptides were extracted for 2 h in a sonicating water bath. After concentration, the peptides were resolved using a Relasil C18 column with a guard pre-column packed with AX-300 on a Michrom HPLC system. Peptides were sequenced at the low picomole level using a modified ABI 477a sequencer employing fast cycle chemistry (18).

Preparation of Recombinant Baculovirus

The coding sequence for bovine CK1alpha (a kind gift from Dr. Melanie Cobb, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX) was amplified using the polymerase chain reaction (PCR) using primers that inserted a Kozak (GCC ACC) sequence upstream of an epitope, FLAG-TAG (MDYKDDDDK), at the N terminus of CK1alpha (5'-primer, CCCAAGCTTGCCACCATGGACTACAAGGACGACGATGACAAGATGGCGAGCAGCAGCGGC; 3'-primer, CCCGGATCCTTAGAAACCTGTGGGGGTTTGGGC). The resulting polymerase chain reaction product was cloned into the XbaI-BamHI sites of pVL1392 (Pharmingen). Recombinant baculovirus was generated using the BaculoGold system (Pharmingen).

The coding sequence for the m3-muscarinic receptor was cloned into the BamHI-EcoRI sites in pVL1393. Recombinant baculovirus was then generated using the BaculoGold system. This baculovirus was used in control infections. (Note that controls were also run using non-infected Sf9 cells with similar results to cells infected with control virus. In this paper experiments with control infected cells are reported.)

Purification of CK1alpha from Infected Sf9 Cells

Sf9 cells (1 liter at 1 × 106 cells/ml) were infected with baculovirus containing the CK1alpha or control baculovirus (1-4 plaque-forming units/cell). After 4 days the cells were harvested and resuspended in 45 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 100 µg/ml benzamine, 100 µg/ml iodoacetamide). After 10 min on ice the cells were homogenized. The homogenate was centrifuged at 20,000 × g for 15 min and a high speed supernatant fraction (S200 fraction) obtained by further centrifugation at 200,000 × g for 35 min. The sample was applied to a 1-ml Resource S (Pharmacia) column which was eluted using a linear gradient of 0-1.0 M NaCl over 20 bed volumes (flow rate = 2 ml/min). 1-ml fractions were collected. The kinase activity eluted as a single peak at ~0.3 M NaCl. These fractions were combined and passed through a 1-ml heparin-Sepharose column equilibrated with 0.32 M NaCl. The column was eluted using a linear gradient of 0.32-1.75 M NaCl over 15 ml. The kinase activity eluted as a single peak at ~0.94 M NaCl. The peak activity was dialyzed against 0.3 M NaCl in TE buffer and stored at 4 °C. The kinase in this preparation was ~4 ng/µl (0.1 pmol/µl), and represents ~3.5% of the total protein in the fraction.

Control purification protocol involved identical purification steps from Sf9 cells infected with the m3-muscarinic receptor baculovirus. The total protein in fractions obtained from the heparin purification of the control and CK1alpha -infected cells were very similar, 100 µg/ml total protein in the control, and 112 µg/ml from the CK1alpha -infected cells.

Assay for Muscarinic Receptor Kinase and Casein Kinase Activity

Assay for the muscarinic receptor kinase involved using the muscarinic receptor fusion protein Ex-m3 as a substrate for the kinase (17).

Samples from chromatography fractions (10 µl) or from supernatant fractions from infected Sf9 cells were incubated with purified Ex-m3 (3.5 µg) or dephosphorylated alpha -casein (15 µg) in kinase buffer (20 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, pH 7.4) containing 50 µM [gamma -32P]ATP (0.4-1.0 cpm/fmol) for 10 min at 37 °C (final volume = 110 µl). Where Ex-m3 was used the reaction was terminated by addition of 1 ml of ice-cold TE buffer. Glutathione-Sepharose (20 µl, Pharmacia) was added and collected by centrifugation (13,000 × g, 10 s) and washed twice with 1 ml of TE buffer. Bound fusion protein was dissociated by boiling in 2 × SDS-PAGE sample buffer (20 µl).

Where alpha -casein was used, the reaction was stopped by the addition of 100% trichloroacetic acid (11 µl). The precipitated proteins were pelleted by centrifugation in a Microfuge for 10 min at 13,000 × g. The protein pellet was washed with acetone (-20 °C) and resuspended in 20 µl of 2 × SDS-PAGE sample buffer.

The proteins from the Ex-m3 or casein assay were resolved by 12% SDS-PAGE. To ensure the equal recovery of the protein substrates and to confirm their relative positions, gels were stained with Coomassie Blue. Gels were then dried and autoradiographs obtained and/or bands corresponding to the peptide substrates were excised and counted.

Phosphorylation of m3-muscarinic Receptors in Membrane Preparations from CHO-m3 Cells

Membrane phosphorylations were carried out as described previously (17). Briefly, crude CHO-m3 cell membranes were prepared and resuspended in kinase buffer at 1 mg of protein/ml. 50 µl of membranes (~0.1 pmol of receptor) were used in a phosphorylation reaction mixture that contained final concentrations of 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM EGTA, 100 µM [gamma -32P]ATP (1-4 cpm/fmol ATP), ± 1 mM carbachol and ±20 µM atropine. To this reaction mixture 10 µl of partially purified CK1alpha (0.5-1 pmol) or control extract was added. Total volume was 100 µl. Reactions were started by the addition of ATP and continued at 32 °C for 10 min. Reactions were stopped by centrifugation at 13,000 × g for 30 s. The supernatant was removed by aspiration and membranes solubilized with 1 ml of solubilization buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 500 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate) for 30 min on ice. m3-Muscarinic receptors were then immunoprecipitated with a specific antiserum (332) as described previously (15).

Phosphorylation of Rod Outer Segment Membranes

Urea-treated bovine rod outer segment membranes (30 pmol of rhodopsin/reaction; a kind gift from Dr. Martin Lohse, Institute of Pharmacology and Toxicology, University of Wurzburg, Versbacher Strasse 9, D-97078 Wurzburg, Germany) were added to kinase buffer containing 50 µM ATP (1 cpm/fmol). To this partially purified CK1alpha (0.3 pmol), purified bovine beta -ARK (0.3 pmol; a gift from Dr. Martin Lohse), or control extract were added. Total volume was 30 µl. The above reagents were combined at 4 °C (where necessary under a safe light). Reactions were started by placing the tubes in a water bath at 32 °C either under a safe light or in room fluorescent light for a given time period. Reactions were stopped by addition of 10 µl of 2 × SDS-PAGE sample buffer. Proteins were then resolved on a 12% gel. Gels were stained with Coomassie Blue, dried, and autoradiographs obtained. Bands corresponding to rhodopsin were excised and counted.

Miscellaneous Procedures

Determination of relative intensities of phosphorylated bands was carried out using a Bio-Rad GS 670 densitometer. Western blotting of whole cell extracts from Sf9 cells used a commercially available antiserum (FLAG-M2, Kodak) that recognizes the FLAG epitope cloned onto the N terminus of CK1alpha .


RESULTS

Amino Acid Sequence Analysis

Proteolytic fragments of MRK purified from porcine brain were isolated by high performance liquid chromatography. The amino acid sequences derived from two peptides were determine to be: 1, WYGQEK; and 2, IEYVHTK. These sequences matched exactly to sequences found exclusively within CK1alpha (peptide 1, Trp78-Lys83; and peptide 2, Ile124-Lys130). Furthermore, the predicted molecular mass of CK1alpha is 37.5 kDa, which corresponds closely with the ~40-kDa mass suggested for MRK (17).

Co-purification of MRK Activity and Casein Kinase Activity

To determine if the kinase we previously defined as MRK was CK1alpha , samples from fractions obtained from the purification of MRK were assayed for both MRK activity, which is defined by the ability of fractions to phosphorylate the bacterial fusion protein Ex-m3 (glutathione S-transferase:m3-third intracellular loop; see "Experimental Procedures"), and casein kinase activity. Fractions from the final two column purification steps, namely the Resource S and heparin-Sepharose fractionation (see Ref. 17), were analyzed. The casein kinase activity was found to elute from the Resource S (data not shown) and heparin-Sepharose columns in an identical manner to MRK activity (Fig. 1). The peak of MRK and casein kinase activity co-eluted from the Resource S and heparin-Sepharose columns at ~0.37 and ~0.87 M NaCl, respectively.


Fig. 1. Co-purification of MRK activity and casein kinase activity. Fractions from the heparin-Sepharose chromatography column step in the purification of porcine brain MRK were tested for MRK activity (i.e. the ability to phosphorylate the m3-muscarinic receptor fusion protein, Ex-m3) and casein kinase activity. The top panel shows an autoradiograph of fractions tested for phosphorylation of the fusion protein Ex-m3. The bottom panel shows the same fractions used to phosphorylate casein (the data are expressed as counts/10 min incorporated into casein).
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Expression of Recombinant CK1alpha in Sf9 Cells

Attempts to express recombinant bovine CK1alpha in mammalian cells (COS-7, HEK 294, and CHO cells) have not proven successful. The reason for this is unclear and is currently under investigation. To obtain a source of recombinant CK1alpha , we turned to the insect cell baculovirus expression system.

Recombinant CK1alpha was expressed in infected Sf9 cells as determined by Western blotting of whole cells using an antiserum against the FLAG-TAG epitope engineered onto the N terminus of CK1alpha (Fig. 2A). The activity of the recombinant kinase was confirmed by an increase in casein kinase activity in cytosolic extracts from CK1alpha -infected Sf9 cells compared with the control cell infection (Fig. 2B). Correlating with an increase in casein kinase activity was an increase in the ability of the cell extract obtained from the CK1alpha -infected cells to phosphorylate the m3-muscarinic receptor fusion protein, Ex-m3 (Fig. 2B).


Fig. 2. Identification of recombinant CK1alpha expression and kinase activity from infected Sf9 cells. A, Western blot of whole cell extract (30 µg of protein/lane) from cells infected with control baculovirus or CK1alpha baculovirus. The antiserum used was a commercially available monoclonal antiserum against a FLAG epitope engineered on the N terminus of CK1alpha . B, high speed supernatant extracts from Sf9 cells infected with control virus or CK1alpha virus were tested for the ability to phosphorylate the m3-muscarinic receptor fusion protein Ex-m3 (3.5 µg) and casein (15 µg). The relative positions of casein and Ex-m3 as determined by Coomassie Blue staining are indicated. C, high speed supernatant extracts from Sf9 cells infected with control virus or CK1alpha virus were fractionated on a Resource S column and fractions tested for the ability to phosphorylate Ex-m3 and casein.
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To confirm that the recombinant CK1alpha contained in infected cell extracts was able to phosphorylate both casein and Ex-m3, the cell extracts were fractionated on a Resource S column. The peak of casein kinase activity contained in CK1alpha -infected cells co-eluted with the peak of MRK activity (Fig. 2C). The peak of recombinant kinase activity eluted at ~0.3 M NaCl. Note, the presence of endogenous casein kinase activity is evident in the control fractionation (Fig. 2C). MRK activity and casein kinase activity also co-eluted during heparin-Sepharose chromatography (data not shown).

Phosphorylation of m3-Muscarinic Receptors and Rhodopsin by Recombinant CK1alpha

Previous studies have demonstrated that membranes from CHO-m3 cells contained an endogenous protein kinase able to phosphorylate the m3-muscarinic receptor in an agonist-dependent manner (17). In these earlier studies, addition of cerebellum-derived MRK to the CHO-m3 membrane preparation resulted in an enhancement of agonist-sensitive m3-muscarinic receptor phosphorylation (17). A parallel experiment was conducted here using partially purified extracts from infected Sf9 cells as the source of exogenous kinase. Fig. 3 shows that in the presence of control extract, purified on the Resource S column, the m3-muscarinic receptor (~0.1 pmol of receptor/reaction) contained in CHO-m3 membranes undergoes agonist-sensitive phosphorylation which can be inhibited by the muscarinic antagonist atropine. Addition of Resource S purified extract from Sf9 cells infected with CK1alpha baculovirus (0.5-1 pmol of CK1alpha /reaction) resulted in an ~2.3-fold increase in agonist-sensitive m3-muscarinic receptor phosphorylation with no significant change in the basal phosphorylation of the receptor (Fig. 3). Furthermore, the increase in agonist-sensitive muscarinic receptor phosphorylation, mediated by CK1alpha , was inhibited by the antagonist atropine (Fig. 3).


Fig. 3. Phosphorylation of the m3-muscarinic receptor by CK1alpha . Membranes prepared from CHO-m3 cells expressing the m3-muscarinic receptor (50 µg of protein/~0.1 pmol of receptor) were reconstituted in a kinase assay with Resource S purified CK1alpha (0.5-1 pmol kinase) derived from CK1alpha -infected Sf9 cells, or control sample purified from Sf9 cells infected with control virus. The receptors were stimulated with 1 mM carbachol or 1 mM carbachol plus 20 µM atropine. Following a 10-min incubation at 32 °C the membranes were solubilized and the receptor immunoprecipitated and the isolated proteins resolved by 8% SDS-PAGE. The positions of molecular weight markers (in kDa) are indicated. The data shown are representative of five experiments carried out in duplicate.
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To further test the ability of CK1alpha to phosphorylate GPCR's, rhodopsin, contained in urea-treated rod outer segments, was used as substrate for CK1alpha . Heparin-Sepharose-purified CK1alpha (~300 fmol) was incubated with rod outer segment membranes (containing rhodopsin at 30 pmol/reaction) either under a safe light or under room fluorescent lights. Rhodopsin phosphorylation was observed only in the extract obtained from cells infected with CK1alpha baculovirus and not from control extracts (Fig. 4A). The CK1alpha extract did phosphorylate rhodopsin under dark conditions and this phosphorylation was enhanced in the presence of light (118 fmol of phosphate incorporated/5-min reaction) (Fig. 4A). In comparison, beta -ARK (~300 fmol/reaction) did not phosphorylate rhodopsin in the absence of light but in the presence of light rhodopsin was phosphorylated to a similar extent as that seen for light-mediated CK1alpha phosphorylation (122 fmol of phosphate incorporated/5-min reaction) (Fig. 4A). The time course for CK1alpha -mediated phosphorylation of rhodopsin was found to be similar to the time course for beta -ARK-mediated phosphorylation (Fig. 4B).


Fig. 4. Phosphorylation of rhodopsin by CK1alpha and beta -ARK. Urea-treated rod outer segment membranes (30 pmol of rhodopsin) were incubated with heparin-Sepharose purified CK1alpha (300 fmol) derived from Sf9 cells infected with CK1alpha , control sample purified from Sf9 cells infected with control virus, or beta -ARK (300 fmol) purified from bovine cerebral cortex. The reactions were stopped by adding 2 × SDS-PAGE buffer and the proteins resolved by 12% SDS-PAGE. The position of rhodopsin, as determined by staining the gels with Coomassie Blue, is indicated. A, the phosphorylation reactions were incubated at 32 °C for 5 min either in the presence or absence of room fluorescent lights. The data shown are representative of four experiments. B, time course of rhodopsin phosphorylation mediated by beta -ARK and CK1alpha under room lights. The control lane (Cnt) is a 40-min reaction of rod outer segment membranes under phosphorylation conditions in the absence of added kinase. The data shown are representative of two experiments.
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DISCUSSION

The present study has revealed that the 40-kDa protein kinase purified previously from porcine cerebellum, called MRK due to its ability to phosphorylate the m3-muscarinic receptor in an agonist-dependent manner (17), can be identified as CK1alpha . These data, together with the ability of recombinant CK1alpha to phosphorylate the m3-muscarinic receptor and rhodopsin in a stimulus-dependent manner, indicate that CK1alpha offers an alternative protein kinase pathway, from that of beta -ARK and the other GRKs, for the agonist-sensitive phosphorylation and the potential regulation of GPCR's.

Due to the presence of endogenous receptor kinase activity in membranes from CHO-m3 cells, the enhancement of muscarinic receptor phosphorylation observed in the presence of CK1alpha may be due to an activation of the endogenous kinase rather than a direct phosphorylation of the receptor. This, however, appears unlikely since CK1alpha is also able to phosphorylate rhodopsin in a stimulus-dependent manner, in urea-treated rod outer segment membranes where there is no endogenous kinase activity. These data also suggest that like beta -ARK, CK1alpha acts predominantly on the activated form of the receptor. This conclusion is supported by dose-response analysis of m3-muscarinic receptor phosphorylation in intact CHO-m3 cells which have demonstrated a close correlation between receptor occupancy and receptor phosphorylation (19). Furthermore, the ability of CK1alpha to mediate light-dependent phosphorylation of rhodopsin suggests that the substrate specificity of CK1alpha is broader than just the m3-muscarinic receptor. Experiments are presently in progress to test the receptor-substrate specificity of CK1alpha against a range of cyclase- and PLC-coupled receptors.

Casein kinase activity was one of the first protein kinase activities identified in mammalian cells and is attributed to two enzymes called casein kinase I and casein kinase II (20, 21). The true "biological" casein kinase responsible for phosphorylating casein in mammary glands is a transmembrane protein kinase of the Golgi apparatus that bears no relationship to casein kinase I or II. In this regard casein kinase I and II are misnomers since the biological substrates for these kinases is not casein. The casein kinase I gene family consists of at least six members; CK1alpha , CK1beta (22), CK1gamma 1-3 (23), and CK1delta (24). A number of in vitro substrates for this kinase family have been identified including: glycogen synthase, SV40 large T antigen (20), p53 tumor suppressor protein (25), and DARPP-32 (26). However, it is not clear which are the biologically relevant substrates. The present study is the first to demonstrate that CK1alpha is able to phosphorylate GPCR's in a stimulus-dependent manner.

The broad receptor substrate specificity of beta -ARK (5, 27) and other members of the GRK family (11-14), as determined in reconstituted systems, has implicated this protein kinase family in the phosphorylation of PLC-coupled receptors. Furthermore, expression of the dominant negative mutant of beta -ARK inhibits agonist-sensitive phosphorylation of PLC-coupled alpha 1B-adrenergic receptors (7) and type 1A angiotensin II receptors (8). However, recent studies have indicated that the intracellular environment following activation of PLC-coupled receptors would not favor the membrane translocation of beta -ARK and other GRK's. It is now clear that beta -ARK activity in reconstituted systems is completely dependent on the presence of the phospholipid, PIP2 (28). Furthermore, translocation of beta -ARK to the plasma membrane is dependent on the synergistic action of G-protein beta gamma -subunits and PIP2 at a site within the pleckstrin homology domain at the C terminus of beta -ARK (28, 29). PIP2 is also involved in the anchoring of GRK-4, GRK-5, and GRK-6 to the plasma membrane by interacting with sites at the C terminus (30). Therefore, receptors that regulate the level of PIP2 could have a profound influence on the activation and translocation of beta -ARK and other members of the GRK family. Stimulation of the m3-muscarinic receptor in CHO-m3 cells3 and in the human neuroblastoma cell line SH-SY5Y cells results in a 80% fall in the levels of PIP2 within the first 10 s of agonist stimulation and this is sustained for 10 min in the presence of agonist (31, 32). Similar rapid falls in the level of PIP2 have been reported to occur following stimulation of m1-muscarinic (33, 34), vasopressin (35), and thrombin receptors (36, 37). PLC-coupled receptor activation may, therefore, discourage membrane translocation/association of the GRK's by depleting PIP2.

In addition, recent studies indicate that calmodulin, in a calcium-dependent manner, can inhibit the association of beta -ARK and GRK-5 with the plasma membrane (38, 39). beta -ARK translocation is inhibited by the ability of calcium/calmodulin to compete for the binding of beta gamma -subunits, and GRK-5 association with the membrane is disrupted by a direct interaction with the kinase (38). Therefore, PLC-coupled receptors, by virtue of their ability to mobilize intracellular calcium stores and mediate calcium entry across the plasma membrane, could again discourage GRK activity.

Overall, it could be anticipated that the intracellular environment following signal transduction via PLC-coupled receptors would not be conducive for GRK translocation and receptor phosphorylation. We would suggest, on the basis of the present study, that CK1alpha offers an attractive alternative route for the phosphorylation of PLC-coupled receptors. In this regard it is interesting to note that casein kinase I isolated from erythrocytes is inhibited by PIP2 (40). Therefore, in contrast to the GRK's, it is possible that PLC-coupled receptors may increase the activity of CK1alpha by the hydrolysis of membrane PIP2. Other regulatory features may also be important such as autophosphorylation which is reported to occur on both tyrosine and serine residues on CK1alpha (41) classifying this kinase as a dual kinase. We have identified autophosphorylation of MRK purified from porcine brain (17) and are presently in the process of determining if autophosphorylation plays any regulatory role in CK1alpha -mediated receptor phosphorylation. Studies are also underway to determine the mechanism of membrane association and the receptor substrate specificity of CK1alpha .


FOOTNOTES

*   This research is supported in part by Wellcome Trust Grants 047600/96 and 16895/96.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Wellcome Trust Senior Research Fellow in Biomedical Science. To whom correspondence should be addressed: Dept. of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN, UK. Tel.: 116-2522935; Fax: 116-2523996; E-mail: TBA{at}le.ac.uk.
1   The abbreviations used are: GPCR, G-protein-coupled receptor; CK1alpha , casein kinase 1alpha ; CHO, Chinese hamster ovary; MRK, muscarinic receptor kinase; PLC, phospholipase C; beta -ARK, beta -adrenergic receptor kinase; GRK, G-protein coupled receptor kinase; PIP2, phosphoinositide 4,5-bisphosphate; Sf9 cells, Spodoptera frugiperda cells; PAGE, polyacrylamide gel electrophoresis.
2   beta -ARK-1 and beta -ARK-2 are also known as GRK-2 and GRK-3, respectively.
3   A. B. Tobin, R. A. Challiss, and S. R. Nahorski, unpublished data.

ACKNOWLEDGEMENTS

We thank Dr. Melanie Cobb for providing the cDNA for casein kinase 1alpha , Dr. Martin Lohse for providing purified beta -adrenergic receptor kinase and bovine rod outer segment membranes, and Dr. Justin Hsuan (Ludwig Institute for Cancer Research, London, UK) for help with amino acid sequencing. We also thank Dr. Gary Willars and Dr. Jonathan Blank for many fruitful discussions.


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