(Received for publication, November 28, 1995)
From the
The apical membrane of intestinal epithelial cells harbors a
unique isozyme of cGMP-dependent protein kinase (cGK type II) which
acts as a key regulator of ion transport systems, including the cystic
fibrosis transmembrane conductance regulator (CFTR)-chloride channel.
To explore the mechanism of cGK II membrane-anchoring, recombinant cGK
II was expressed stably in HEK 293 cells or transiently in COS-1 cells.
In both cell lines, cGK II was found predominantly in the particulate
fraction. Immunoprecipitation of solubilized cGK II did not reveal any
other tightly associated proteins, suggesting a membrane binding motif
within cGK II itself. The primary structure of cGK II is devoid of
hydrophobic transmembrane domains; cGK II does, however, contain a
penultimate glycine, a potential acceptor for a myristoyl moiety.
Metabolic labeling showed that cGK II was indeed able to incorporate
[H]myristate. Moreover, incubation of cGK
II-expressing 293 cells with the myristoylation inhibitor
2-hydroxymyristic acid (1 mM) significantly increased the
proportion of cGK II in the cytosol from 10 ± 5 to 35 ±
4%. Furthermore, a nonmyristoylated cGK II Gly
Ala
mutant was localized predominantly in the cytosol after transient
expression in COS-1 cells. The absence of the myristoyl group did not
affect the specific enzyme activity or the K
for cGMP and only slightly enhanced the thermal stability of
cGK II. These results indicate that N-terminal myristoylation fulfills
a crucial role in directing cGK II to the membrane.
Cyclic GMP-dependent protein kinases (cGK) ()play an
important role in cGMP-mediated signaling pathways which are triggered
by various hormones and neurotransmitters including nitric oxide (NO),
natriuretic peptides, and guanylin(1, 2) . In
mammalian tissues, two types of cGK have been identified. Type I cGK,
consisting of
and
isoforms, possible splice variants of a
single gene, is more generally expressed and acts as a key regulator of
cardiovascular homeostasis(1, 2) . In contrast, type
II cGK was described originally as an intestine-specific
form(3) . Molecular cloning demonstrated that cGK II is indeed
a distinct gene product expressed predominantly in epithelial cells of
the intestine(4) , although its mRNA was also detected in
kidney and brain(4, 5, 6) . Its widespread
distribution in various areas of the brain suggests an important role
of cGK II in the NO/cGMP signaling in the central nervous
system(6) .
In the intestine, cGMP is involved in the regulation of ion and water transport. It inhibits the uptake of NaCl and stimulates the secretion of chloride by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel which is mutated in CF patients (7, 8) . Heat-stable enterotoxins increase cGMP and elicit a severe secretory diarrhea by activating an intestine-specific isoform of guanylyl cyclase(9) . Guanylin, a heat-stable enterotoxin-like peptide, may function as the physiological activator of the cGMP-mediated signaling pathway in intestine(10) . Recent electrophysiological and localization studies suggest a key role for cGK II as a mediator of the cGMP-provoked intestinal chloride secretion(8, 11, 12) .
The positioning of cGK II in the apical membrane of the enterocyte, facilitating its interaction with transport systems in the same compartment, may contribute importantly to its efficacy as an ion transport regulator. However, the mode of cGK II membrane anchoring has not been elucidated. The primary sequence of cGK II, like the sequence of type I cGK, a known cytosolic and/or peripheral protein, reveals no obvious hydrophobic transmembrane domains(2, 4) . Instead, the membrane localization of cGK II might be determined by its association with a distinct anchor protein. A family of anchor proteins exists for the regulatory subunit of cAMP-dependent protein kinase (cAK) (13) , and the intermediate filament protein vimentin may function as a receptor for cGK I(14) . Alternatively, cGK II itself may acquire hydrophobic properties through the attachment of a lipid moiety. A number of proteins involved in signal transduction, e.g. Src, Ras, and trimeric G-proteins require lipid modifications like myristoylation, palmitoylation, or isoprenylation for membrane binding (15) . Type II cGK lacks a consensus sequence for isoprenylation, but the penultimate glycine in cGK II might serve as an acceptor for a myristoyl group(15) . We report here that recombinant cGK II expressed in HEK-293 and COS-1 cells is indeed myristoylated and that this lipidation is required for its attachment to the membrane.
Protein kinase activity was determined by incubating
samples (5-10-µl aliquots containing 5-10 µg of
protein) at 30 °C for 10 min in 50 µl of 20 mM Tris-HCl, pH 7.4, 10 mM MgCl, 5 mM
-mercaptoethanol, 0.1 mM 3-isobutyl-1-methylxanthine, 200 nM protein kinase A
inhibitor, 0.1 mM [
-
P]ATP (200
cpm/pmol), and a 0.1 mg/ml concentration of a cGK-specific substrate
peptide 2A3 (RRKVSKQE) as described(17) . To reduce high
nonspecific background phosphorylation and to facilitate the exposure
of membrane-bound cGK II to the exogenous substrate, 1% Triton X-100
and 0.5 M NaCl were added to all fractions prior to assay.
High speed centrifugation of homogenates of HEK 293 cells
stably expressing rat cGK II resulted in recovery of 90-95% of
expressed cGK II in the membrane fraction as detected by Western
blotting (cf. Fig. 3). The enzyme could be released
from the membranes by a combination of detergent (1% Triton X-100) and
high salt (0.5 M NaCl) but not by detergent (Fig. 1) or
high salt alone (not shown). This suggests that recombinant cGK II is
attached both to the membrane and the cytoskeleton in HEK 293 cells,
resembling native cGK II in rat or pig intestinal brush-border
membranes(3) , and argues against the need for an
intestine-specific factor for targeting the enzyme to the membrane.
Conceivably, membrane attachment could occur through cGK II binding to
a more ubiquitous anchor. However, we did not detect any proteins
associated with cGK II in immunoprecipitates from HEK 293 cells
metabolically labeled with [S]methionine (Fig. 2A). Furthermore, native cGK II still displays
hydrophobic properties after its solubilization and
purification(3) . Therefore, the membrane binding of cGK II is
unlikely to depend solely on its association with anchor proteins.
Figure 3:
2-Hydroxymyristic acid promotes cytosolic
localization of cGK II by inhibiting myristoylation. HEK 293 cells
stably transfected with pRc/CMV-cGK II were incubated for 48 h with or
without 1 mM myristoylation inhibitor 2-hydroxymyristic acid (HMA). Subsequently, cells were either homogenized and cGK II
was determined in the homogenate (hom), cytosolic (cyt), and membrane (mem) fractions by immunoblotting (lower panel) or cells were incubated for an additional 4 h
with [H]myristate prior to homogenization and
separation into a cytosolic or membrane fractions. Incorporation of
[
H]myristate into cGK II was detected by
autoradiography (20 days) following immunoprecipitation of cGK II and
subsequent separation on 7.5% SDS-PAGE (upper
panel).
Figure 1:
Western blot demonstrating that
solubilization of cGK II requires high salt in combination with Triton
X-100. HEK 293 cells stably transfected with pRc/CMV-cGK II were
homogenized and centrifuged at 20,000 g to obtain a
heavy membrane fraction which was then incubated with 1% Triton X-100
for 5 min at 0 °C in the presence of various concentrations of NaCl
as indicated. Subsequently, samples were removed before and after
centrifugation (15 min, 20,000
g) for detection of cGK
II present in the total extract (tot) and supernatant (sup) and pellet (pel) fractions by immunoblotting.
The results shown are representative of three
experiments.
Figure 2:
Autoradiogram of cGK II immunoprecipitated
from HEK 293 cells metabolically labeled with
[S]methionine or
[
H]myristate. A, HEK 293 cells
mock-transfected or stably transfected with pRc/CMV-cGK II were
incubated for 4 h with [
S]methionine and lysed
with 1% Triton X-100 and 0.5 M NaCl. cGK II was
immunoprecipitated with a specific antibody and analyzed using 10%
SDS-PAGE and autoradiography (4 days). B, HEK 293 cells stably
transfected with pRc/CMV-cGK II were incubated for 4 h with
[
H]myristate, lysed with 1% Triton X-100 and 0.5 M NaCl, and analyzed by 10% SDS-PAGE prior to (lys)
or after immunoprecipitation (IP) with cGK II antibody. Label
incorporated was detected by autoradiography (30
days).
The alternative possibility that cGK II may be lipid-modified was
tested by incubating HEK 293 cells stably expressing cGK II with
[H]myristate. As shown in Fig. 2B, a protein with the correct M
(86,000) for cGK II was the major radioactively labeled protein
in a total lysate. This myristoylated protein could be precipitated
with a cGK II specific antibody, further confirming its identity as cGK
II. No evidence was obtained for a second covalent modification by
palmitoylation, since no radioactivity was detected in cGK II
immunoprecipitated from transfected 293 cells metabolically labeled
with [
H]palmitate (not shown).
In order to
investigate whether myristoylation plays a role in the membrane
attachment of cGK II, the cGK II-expressing 293 cells were incubated
for 48 h with a 1 mM concentration of the myristoylation
inhibitor 2-hydroxymyristic acid(18) . Inhibitor treatment
significantly increased the amount of cGK II recovered in the cytosolic
fraction (35 ± 4%) compared with that of control cells (10
± 5%) (n = 5 for each group; Fig. 3).
Metabolic labeling of the cells with [H]myristate
in the presence of 2-hydroxymyristic acid showed that the form of cGK
II accumulating in the cytosol was nonmyristoylated (Fig. 3),
indicating that shift in cGK II topology from the membrane to the
cytosol was indeed due to inhibition of myristoylation. High salt (0.5 M NaCl) had no effect on the amount of cGK II recovered in the
cytosol fraction of cells preincubated with 2-hydroxymyristic acid,
indicating that the fraction of cGK II which remained bound to the
membrane was anchored by hydrophobic interactions.The lack of a
complete reallocation of cGK II to the cytosol most likely reflects an
incomplete blockade of myristoylation by the inhibitor, since
[
H]myristate incorporation was still detectable
in the membrane-bound cGK II pool after treatment with
2-hydroxymyristic acid (Fig. 3).
To further investigate the
membrane binding properties of nonmyristoylated cGK II, we mutated the
penultimate glycine to an alanine. As shown in Fig. 4A,
this G2A mutant, in contrast to wild type cGK II, was unable to
incorporate [H]myristate after transient
expression in COS-1 cells, although wild type and mutant cGK II were
expressed to similar levels as detected by immunoblotting (Fig. 4B). As observed earlier in HEK 293 and
intestinal cells, wild type cGK II was predominantly bound to the
membrane fraction of COS-1 cells (Fig. 4C). The removal
of Gly
, however, resulted in the shift of a major
proportion of cGK II from a membrane to a cytosolic localization (Fig. 4C), providing further evidence that
myristoylation is required for membrane binding of cGK II. The small
amount of the G2A-cGK II mutant recovered in the membrane fraction most
plausibly represents cytosolic enzyme entrapped in incompletely lysed
cells or in vesiculated membranes.
Figure 4:
Localization of the nonmyristoylated cGK
II mutant G2A in COS-1 cells. COS-1 cells were transiently transfected
with pRc/CMV-cGK II (wt) or a mutant-cGK II in which Gly was changed to Ala (G2A). A and B, 2 days after
transfection, cells were labeled with
[
H]myristate and cGK II was immunoprecipitated
and analyzed by SDS-PAGE followed by autoradiography (20 days) (A) or immunoblotting (B). C, 2 days after
transfection, cGK II was determined in the homogenate (H),
cytosolic (C), and membrane (M) fractions by
immunoblotting.
Paradoxically, mouse brain cGK
II, which displays a high level of homology to rat intestine cGK
II(4, 5) , was reported to reside predominantly (98%)
in the cytosol when expressed in COS-1 cells(5) . This
subcellular localization of mouse cGK II, however, was based on
measurements of protein kinase activity rather than on immunological
detection. Therefore, we compared the kinase activities of rat cGK II
and the G2A mutant in the homogenate, cytosolic, and membrane fraction
of COS-1 cells. As shown in Fig. 5, only a small proportion of
the cGK II activity was present in the cytosol compared to the membrane
fraction of COS-1 transfected with wild type cGK II, whereas most of
the activity of the nonmyristoylated G2A mutant was cytosolic. The
kinase activities correlated well with the levels of immunodetectable
cGK II in the various fractions (compare Fig. 4and 5),
indicating that the specific activity of the myristoylated and
nonmyristoylated forms of cGK II are not substantially different.
Likewise, the absence of a myristoyl group did not alter the K for cGMP (0.7 µM for both wild type
cGK II and the G2A mutant; Fig. 6). The K
for cGMP observed here for rat cGK II resembles the K
(0.3 µM) reported for mouse brain
cGK II(5) . These data therefore failed to provide a clear
explanation for the difference in localization found for rat and mouse
cGK II transiently expressed in COS-1 cells. A species difference in
cGK II membrane anchoring properties was also considered, but found
unlikely because: (i) the first nine amino acids of mouse brain and rat
intestine cGK II, which cover the region normally containing the
myristoylation sequence are identical, and (ii) native cGK II from rat
and mouse intestine as well as from rat brain were found to be largely
membrane-associated (data not shown).
Figure 5:
Subcellular distribution of the kinase
activity of cGK II and the nonmyristoylated cGK II mutant G2A. COS-1
cells were transiently transfected with either pRc/CMV-cGK II (wt), a cGK II mutant in which Gly was changed to
Ala (G2A), or the pRc/CMV vector. Two days after transfection,
cells were harvested, homogenized, and fractionated. Kinase activity in
the presence or absence of cGMP (5 µM) was determined
after solubilization of cGK II with 1% Triton and 0.5 M NaCl,
as described under ``Experimental Procedures.'' cGK II kinase
activity was expressed per mg of homogenate protein and corrected for
basal activity found in cells transfected with the pRc/CMV vector. The
basal kinase activities of samples from mock-transfected COS-1 cells
were 92, 94, 13, 17, 61, and 58 pmol/min, respectively, for the
homogenate -/+-cGMP, the cytosol -/+-cGMP, and
the membrane fraction -/+-cGMP. Data are expressed as means
± S.E. of three experiments.
Figure 6:
Activation of cGK II and the
nonmyristoylated cGK II mutant G2A by cGMP. COS-1 cells were
transiently transfected with either pRc/CMV-cGK II (WT;
), a cGK II mutant in which Gly
was changed to Ala (G2A;
), or the pRc/CMV vector (cont;
).
Two days after transfection, cells were harvested and homogenized.
Kinase activity in the presence of various concentrations of cGMP was
determined in the homogenates after solubilization of cGK II with 1%
Triton and 0.5 M NaCl, as described under ``Experimental
Procedures.'' Data are expressed as means ± S.E. of three
experiments.
While these results indicate
that myristoylation is a prerequisite for membrane binding of cGK II,
additional factors might contribute as well. Some myristoylated
proteins like the catalytic subunit of cAK are soluble(19) , or
reversibly attached to the membrane (e.g. MARCKS; (20) ). Therefore, a second membrane binding motif in
combination with the myristoyl group is thought to be required for
membrane attachment(21) . This second motif was shown to be
either a polybasic region, which interacts with the negatively charged
inner membrane surface, or a second lipid moiety like palmitic
acid(21) . No evidence for palmitoylation of cGK II was
obtained in this study. However, cGK II, similar to cGK I(2) ,
appears to function as a dimer under physiological conditions, ()suggesting that two myristoyl groups per molecule of cGKII
are available for membrane binding. The first three lysines in cGKII
(Lys
, Lys
, Lys
), located at
similar positions as the lysines involved in membrane binding of
Src(21) , may also contribute to the membrane binding.
In addition to its role in membrane anchoring, myristoylation was also reported to enhance the stability of certain proteins. Notably, the myristoylated catalytic subunit of cAK is considerably less temperature-sensitive compared to the nonmyristoylated form(22) . We therefore compared the thermal stability of cGK II and the nonmyristoylated G2A mutant. As shown in Fig. 7, G2A was slightly more stable than wild type cGK II (1.5-2 °C), indicating that myristoylation does not stabilize cGK II. This suggests that the myristoyl group in cGK II, in contrast to its counterpart in the catalytic subunit of cAK(23) , is not involved in intramolecular stabilization, but is fully accessible to membrane interaction.
Figure 7:
Temperature sensitivity of cGK II and the
nonmyristoylated cGK II mutant G2A. COS-1 cells were transiently
transfected with either pRc/CMV-cGK II (wt; ) or with a
cGK II mutant in which Gly
was changed to Ala (G2A;
). Two days after transfection, cells were
harvested and homogenized. The homogenates were incubated for 3 min at
various temperatures prior to determination of kinase activity in the
presence or absence of 10 µM cGMP for 5 min at 30 °C
as described under ``Experimental Procedures.'' The
cGMP-stimulated kinase activity is expressed as a percentage of the
cGMP-stimulated kinase activity in homogenates which were preincubated
for 1 min at 30 °C prior to the kinase assay. Data are expressed as
means ± S.E. of three experiments.
Conceivably, the intrinsic hydrophobicity of
myristoylated cGK II promotes its association with membrane-bound
substrates. Two physiologically relevant substrates of cGK II have been
described so far, i.e. a 25-kDa proteolipid detected in
intestinal brush-border membranes whose function is still
unknown(24) , and the CFTR-Cl channel, which is responsible for
the cGMP-mediated electrogenic chloride secretion in the
intestine(8, 12) . Interestingly, purified cGK II, but
not cGK I, is able to activate the CFTR-Cl channel in excised
membrane patches of cells stably expressing CFTR(12) , whereas
both isotypes phosphorylate immunoprecipitated CFTR in
vitro(12, 25) . Since the N terminus of cGK
I
is acetylated rather than myristoylated(26) , it is
tempting to speculate that the different lipidation and membrane
binding properties of cGK II and I, rather than their substrate
specificities, account for the preferential activation of CFTR by cGK
II. By analogy, modulation of the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate
receptor channel by endogenous cAK was shown to be dependent on the
binding of cAK to an anchoring protein, presumably localized in close
proximity to the channel(27) .
The availability of a soluble, catalytically active, nonmyristoylated mutant version of cGK II may greatly facilitate future studies designed to investigate the putative role of membrane anchoring in cGK II regulation of transport functions by a more direct approach.