(Received for publication, March 31, 1997, and in revised form, June 2, 1997)
From the 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
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 -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 1
(CK1
) based on the following evidence: 1) the amino acid sequence
from two proteolytic peptide fragments derived from purified MRK
corresponded exactly to sequences within CK1
. 2) Casein kinase
activity co-eluted with MRK activity from the final two chromatography
steps in the purification of porcine brain MRK. 3) Recombinant CK1
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 CK1
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 CK1
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 CK1
, and
that CK1
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.
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
-adrenergic receptor where agonist-dependent
phosphorylation and receptor desensitization is mediated by the
receptor-specific kinase,
-adrenergic receptor kinase (
-ARK) (2,
3).
Studies using purified or partially purified receptor preparations
reconstituted in phospholipid vesicles with purified -ARK, have
demonstrated that
-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
-ARK to inhibit endogenous
-ARK activity (6)
has suggested that
-ARK is the endogenous kinase responsible for the
phosphorylation of recombinant PLC-coupled
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
-opioid receptors in HEK 293 cells (9). These studies have indicated
that
-ARK may have a broad receptor substrate specificity that
extends beyond
-adrenergic receptors.
-ARK-1 and
-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
2-and
2-adrenergic, and m2-muscarinic receptors (11-13) and
GRK-6 to phosphorylate
2-adrenergic and m2-muscarinic
receptors, in an agonist-dependent manner (14). Therefore,
it appears that
-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 -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
-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 -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
-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 1 (CK1
), and reveal
the ability of the recombinant kinase to phosphorylate m3-muscarinic
receptors and rhodopsin in an agonist/stimulus-dependent manner.
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 -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.).
The procedure used for purification of MRK from porcine cerebellum has been previously described (17).
Preparation of the Bacterial Fusion Protein Ex-m3Preparation 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 SequencingThe 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 BaculovirusThe coding sequence
for bovine CK1 (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 CK1
(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 CK1Sf9 cells (1 liter at 1 × 106 cells/ml) were infected with
baculovirus containing the CK1 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 CK1-infected cells were very similar, 100 µg/ml
total protein in the control, and 112 µg/ml from the CK1
-infected
cells.
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 -casein (15 µg) in kinase buffer
(20 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, pH 7.4) containing 50 µM
[
-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 -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 CellsMembrane 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
[-32P]ATP (1-4 cpm/fmol ATP), ± 1 mM
carbachol and ±20 µM atropine. To this reaction mixture
10 µl of partially purified CK1
(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).
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 CK1
(0.3 pmol), purified bovine
-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.
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 CK1.
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 CK1 (peptide 1, Trp78-Lys83; and peptide 2, Ile124-Lys130). Furthermore, the predicted
molecular mass of CK1
is 37.5 kDa, which corresponds closely with
the ~40-kDa mass suggested for MRK (17).
To
determine if the kinase we previously defined as MRK was CK1,
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.
Expression of Recombinant CK1
Attempts to
express recombinant bovine CK1 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 CK1
, we turned to the insect cell baculovirus expression
system.
Recombinant CK1 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 CK1
(Fig. 2A). The activity of the
recombinant kinase was confirmed by an increase in casein kinase
activity in cytosolic extracts from CK1
-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 CK1
-infected cells to
phosphorylate the m3-muscarinic receptor fusion protein, Ex-m3 (Fig.
2B).
To confirm that the recombinant CK1 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 CK1
-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).
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 CK1
baculovirus (0.5-1 pmol of CK1
/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 CK1
, was inhibited by the
antagonist atropine (Fig. 3).
To further test the ability of CK1 to phosphorylate GPCR's,
rhodopsin, contained in urea-treated rod outer segments, was used as
substrate for CK1
. Heparin-Sepharose-purified CK1
(~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 CK1
baculovirus and not from
control extracts (Fig. 4A).
The CK1
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,
-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 CK1
phosphorylation (122 fmol of phosphate
incorporated/5-min reaction) (Fig. 4A). The time course for
CK1
-mediated phosphorylation of rhodopsin was found to be similar to
the time course for
-ARK-mediated phosphorylation (Fig.
4B).
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 CK1. These data, together with the ability of recombinant CK1
to
phosphorylate the m3-muscarinic receptor and rhodopsin in a
stimulus-dependent manner, indicate that CK1
offers an
alternative protein kinase pathway, from that of
-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 CK1 may be due to an
activation of the endogenous kinase rather than a direct
phosphorylation of the receptor. This, however, appears unlikely since
CK1
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
-ARK, CK1
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 CK1
to mediate light-dependent phosphorylation of rhodopsin suggests that the substrate specificity of
CK1
is broader than just the m3-muscarinic receptor. Experiments are
presently in progress to test the receptor-substrate specificity of
CK1
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; CK1, CK1
(22),
CK1
1-3 (23), and CK1
(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 CK1
is able to phosphorylate GPCR's
in a stimulus-dependent manner.
The broad receptor substrate specificity of -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
-ARK inhibits agonist-sensitive phosphorylation of PLC-coupled
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
-ARK and other GRK's. It is now clear that
-ARK activity in
reconstituted systems is completely dependent on the presence of the
phospholipid, PIP2 (28). Furthermore, translocation of
-ARK to the plasma membrane is dependent on the synergistic action of G-protein
-subunits and PIP2 at a site within the
pleckstrin homology domain at the C terminus of
-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
-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
-ARK and GRK-5 with the plasma membrane (38, 39).
-ARK
translocation is inhibited by the ability of calcium/calmodulin to
compete for the binding of
-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 CK1 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 CK1
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 CK1
(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
CK1
-mediated receptor phosphorylation. Studies are also underway to
determine the mechanism of membrane association and the receptor
substrate specificity of CK1
.
We thank Dr. Melanie Cobb for providing the
cDNA for casein kinase 1, Dr. Martin Lohse for providing
purified
-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.