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J Biol Chem, Vol. 275, Issue 3, 1565-1569, January 21, 2000
From the § Renal Division, Department of Medicine,
Brigham and Women's Hospital and Harvard Medical School, Boston,
Massachusetts 02115 and Fundación Investigaciones
Biológicas Aplicadas, Vieytes 3103, 7600 Mar del Plata, Argentina
One mechanism utilized by cells to maintain
signaling pathways is to regulate the levels of specific signal
transduction proteins. The compound geldanamycin (GA) specifically
interacts with heat shock protein 90 (hsp90) complexes and has been
widely utilized to study the role of hsp90 in modulating the function
of signaling proteins. In this study, we used GA to demonstrate that
levels of heterotrimeric G Cells respond to a wide range of physical, chemical, and hormonal
stimuli through cell surface receptors that are coupled to
heterotrimeric G proteins.1 G
proteins are composed of G An additional cellular mechanism to regulate signaling pathways is to
control the degradation of individual signaling molecules (6). Lipid
modifications on the N terminus of G Chemicals and Reagents--
GA was kindly provided by the Drug
Synthesis and Chemistry Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer Institute.
Hemagglutinin (HA) antibody (clone SCP-12CA5-J) was purchased from
Berkeley Antibody Co., and anti-heat shock protein 90 (mouse monoclonal, IgM) antibody was purchased from StressGen
(Collegeville, PA). Protease inhibitors N-Ac-LLM, LCN,
and Z-LLL were from Biomol Research Laboratories (Plymouth Meeting,
PA). Chemicals were from Sigma, and chemiluminescence reagents were
from Pierce.
Buffers--
The buffers used were: A, 50 mM
Tris-HCl, pH 7.6, 6 mM MgCl2, 75 mM
sucrose, 1 mM dithiothreitol, 1 mM EDTA, 3 mM benzamidine, 1 µg/ml leupeptin, soy and lima bean
trypsin inhibitors; B, 10 mM Tris-HCl, pH 7.4, 1% Triton
X-100, 0.1% SDS, 1% sodium deoxycholate; C, 1% Triton X-100, 20 mM sodium molybdate.
Cell Culture and Transient Transfections--
PC12 cells were
kindly provided by Dr. Eva Neer (Brigham and Women's Hospital, Boston,
MA) and cultured as described previously (11). G Electrophoresis and Immunoblotting--
Levels of
G Metabolic Labeling Studies and Immunoprecipitations--
PC12
cells and transfected COS cells (at 48 h) were treated with 2 µM GA for 18 h (COS cells) or 24 h (PC12 cells)
and then incubated for 30 min in a methionine- and cysteine-free
medium. Tran35S-labelTM (>1000 Ci/mmol, ICN
Radiochemicals) was added to a final concentration of 200 µCi/ml for
60-90 min and chased with medium containing cold methionine/cysteine
for various lengths of time. For synthesis studies, cells were labeled
at t = 0 and immunoprecipitated at 30-min intervals.
Cells were washed and scraped, and homogenates were prepared as
described above. 35S-Labeled proteins were
immunoprecipitated from homogenates in Buffer B after clearing with
protein A-Sepharose. 12CA5 antibody (1/250 µl) for HA
G In Vitro Translation--
cDNAs for wild-type
G Regulating the levels of signaling proteins is an important
mechanism contributing to the accurate development and maintenance of
signal transduction pathways. hsp90 interacts with several families of
signal transduction proteins including receptor and non-receptor
tyrosine kinases, serine threonine kinases, and mutated p53 (reviewed
in Ref. 16). The compound GA destabilizes the interaction between hsp90
and its associated proteins leading to accelerated degradation and loss
of function. Although G The lower steady state protein levels of G
Degradation of Heterotrimeric G
o Subunits via
the Proteosome Pathway Is Induced by the hsp90-specific
Compound Geldanamycin*
¶,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunits can be regulated through
interactions with hsp90. In a dose-dependent manner, GA
significantly reduced the steady state levels of endogenous
Go expression in two cell lines (PC12 and GH3) and
had a similar effect on Go transiently expressed in COS
cells. Go synthesis and degradation was studied in PC12
cells and in transiently transfected COS cells. 35S
labeling followed by immunoprecipitation demonstrated no effect of GA
on the rate of Go synthesis, but GA accelerated
degradation of Go in both PC12 cells and COS cells. The
use of inhibitors, including lactacystin (a proteosome-specific
inhibitor), suggests that Go is predominantly degraded
through the proteosome pathway. In vitro translated
35S-labeled Go could be detected in hsp90
immunoprecipitates, and this interaction did not require N-terminal
myristoylation. Taken together, these results suggest that
heterotrimeric Go subunits are protected from
degradation by interaction with hsp90 and that the interaction of G
subunits with heat shock proteins may be a general mechanism for
regulating G levels in the cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and G
subunits and are attached to
the plasma membrane through lipid modifications on G and G subunits (reviewed in Ref. 1). Activated receptors induce a conformational change in G that leads to GDP release and GTP binding. GTP-bound G dissociates from G, and both subunits can
interact with a variety of intracellular effectors until the intrinsic
GTPase activity of G hydrolyzes GTP to GDP. Many types of G
protein-coupled receptors and G proteins are expressed within the same
cell, and the mechanisms that generate specific cellular responses are
not well understood. Some specificity lies at the interface of
receptor-G protein and G protein-effector, but in reconstituted systems
multiple G proteins can couple to the same sets of receptors and
effectors (reviewed in Ref. 2). Furthermore, in a single cell type, a
single G subunit can couple to at least three different effector
pathways (3). In the cell, multiple mechanisms are likely to be
important for maintaining signaling specificity, and these include
regulation by modulatory proteins such as receptor kinases (reviewed in
Ref. 4) and RGS (regulators of G protein signaling) proteins (reviewed
in Ref. 5).
subunits are important for
targeting and attachment to the plasma membrane, but the interaction of
G subunits with cytosolic proteins prior to plasma membrane
association have not been defined. The 90-kDa heat shock protein
(hsp90) is a highly conserved protein chaperon representing up to 5%
of total cell protein under non-stress conditions. Interestingly, hsp90
interacts with a diverse group of proteins involved in cellular
signaling that include several families of tyrosine kinases and steroid
hormone receptors. The functional implications for the interaction of
signaling molecules with hsp90 depends upon the protein. Steroid
hormone receptors are stabilized by interaction with hsp90, and this
interaction is necessary for high-affinity ligand binding (7). In
contrast, the membrane-associated tyrosine kinase
pp60v-svc interacts transiently with hsp90
following synthesis until the nascent protein is inserted in the
membrane (8). The role of hsp90 in regulating the functions of some
cellular proteins has been facilitated by the recognition that quinone
ansamycin antibiotics, such as geldanamycin (GA), are highly specific
inhibitors of hsp90-protein complexes (9). The GA-hsp90 complex has
been crystallized and reveals GA binding to a highly conserved pocket
of hsp90 (residues 9-232) (10). Because hsp90 interactions are
important for the proper function of a variety of signaling molecules,
we asked whether G protein subunits could also interact with hsp90.
We used the hsp90-specific compound geldanamycin to address this question, and we found that GA induced a decline in the level of
endogenous Go in PC12 cells, GH3 cells, and transiently
transfected COS cells without affecting the level of other cellular
proteins. The enhanced degradation of Go occurred
predominantly through the proteosome pathway. Furthermore,
immunoprecipitates of hsp90 from in vitro translates of
Go coprecipitated Go, and this
interaction was independent of N-terminal myristoylation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
o
cDNA in Bluescript (12) was amplified by polymerase chain reaction
to generate a blunt C-terminal end that was then cloned into a modified
Bluescript vector containing the HA epitope. The sequence was confirmed
by dideoxynucleotide sequencing. HA Go was cloned into
the eukaryotic expression vector pcDNA3 (Invitrogen) using the
XbaI and ApaI sites. COS cells were cultured and
transfected as described previously using LipofectAMINE (Life
Technologies, Inc.) (13), and 24-48 h after transfection cells were
treated with GA dissolved in dimethyl sulfoxide or with dimethyl
sulfoxide alone under various conditions.
o expression were determined 48-72 h after
transfection of COS cells or from confluent monolayers of PC12 cells and GH3 cells after treatment with GA for specified conditions. Cells
were washed with phosphate-buffered saline, suspended in 300 µl of
cold Buffer A, frozen and thawed 3 times in liquid nitrogen, and passed
15 times through a 27-gauge needle. The homogenates were cleared by
centrifuging at 1,500 rpm for 5 min at 4 °C. Total protein levels
were determined by the Bradford method, and SDS-PAGE sample buffer was
added to the supernatant. Equivalent amounts of total protein were
analyzed by SDS-PAGE, and the level of Go expression was
determined by Western blotting using the anti-HA monoclonal antibody at
a dilution of 1:1000 or an anti-Go antibody (R4) at a
dilution of 1:2000 (courtesy of E. Neer (14)). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies were used at a dilution of 1:10,000, and bands were visualized by chemiluminescence.
o transiently expressed in COS cells (or R4 (1/100
µl) for PC12 cells) was added for 1-4 h at 4 °C followed by
protein A-Sepharose for 1 h. Samples were centrifuged, and the
pellets were washed three times with Buffer A. The immunoprecipitates were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by autoradiography.
o (not HA-tagged) and myristoylation-minus mutant (G1A)
were in vitro translated with [35S]methionine
in a single-step rabbit reticulocyte lysate (Promega) as described
previously (13). The in vitro translation was mixed 1:1 with
2× Buffer C and then divided in half. One tube was incubated with the
anti-hsp90 antibody coupled to protein A-Sepharose (15), and the other
was a control of nonspecific IgM antibody similarly coupled to protein
A-Sepharose. The mixtures were rocked at 4 °C for 1 h,
centrifuged, and washed three times with cold Buffer C. Samples were
eluted with SDS-PAGE sample buffer and analyzed as described above.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunits have been shown to interact with
hsp90 (17), little is known about the interaction of G subunits with
heat shock proteins. To determine whether there is a role for hsp90 in
regulating G subunits, we used GA to characterize Go
protein levels in two different systems: 1) endogenous
Go in neuronal cell lines (PC12 and GH3) and 2)
Go in transiently transfected COS cells. To
facilitate future studies, we were interested in determining whether
the mechanisms of Go degradation were similar in PC12 cells and in a transient expression system. When cells were treated with increasing concentrations of GA (Fig.
1) for 18 h or with 2 µM GA for increasing lengths of time (not shown), there
were dose- and time-dependent decreases in steady state
Go levels for all three cell lines. Interestingly, 2 nM GA treatment caused a noticeable increase in steady
state Go levels (Fig. 1) in all three cell lines. The
explanation for this observation is not immediately apparent but may
relate to compensatory changes in synthesis and/or degradation at low
dose exposure to GA. C-terminal hemagglutinin epitope-tagged
Go was used in the COS cell studies to facilitate
subsequent experiments requiring immunoprecipitations. Transient
expression of Go in COS cells results in over 75% of the protein separating into the particulate fraction (13, 18), an
amount consistent with results in bovine brain (19). In addition, placing the epitope on the C terminus does not affect separation into
soluble and particulate fractions (not shown), and HA Go exchanges guanine nucleotides as determined by a tryptic conformation assay (not shown). Furthermore, the HA epitope on the C terminus of
Gpa1, the yeast G subunit, did not affect the stability of the
protein (6). The closed arrows (Fig. 1) indicate
Go; the open arrowhead indicates a
nonspecific protein present in vector-transfected (PC) control cells
(first lane) and in HA Go-transfected cells. The
intensity of the nonspecific protein does not change with increasing GA
doses, but there is a dose-dependent decrease in steady
state Go levels (Fig. 1, closed arrows). The
time-dependent effects of 2 µM GA were not
apparent until after 3 h, and maximal effects were consistently
seen with 18-24 h of exposure (not shown). Steady state
Go levels were reduced by 78 ± 2% (n = 7) in COS cells and to a similar degree in PC12 and GH3 cells.
View larger version (32K):
[in a new window]
Fig. 1.
GA effects on steady state
G o levels in PC12, GH3, and
transfected COS cells. PC12 (closed circles) and GH3
cells (open circles) were treated with GA (0-20
µM) for 24 h. Equivalent amounts of total protein
were analyzed by Western blotting using a rabbit anti-Go
antibody. COS cells (triangles) were transfected with HA
Go or pcDNA3 cDNAs as described under
"Experimental Procedures." After 36-48 h, cells were incubated
with increasing concentrations of GA for 18 h, and equivalent
amounts of total protein were analzyed by Western blotting using 12CA5
antibody to the HA epitope. Top, autoradiograms for each
cell line. Lane 1, COS cells only (transfected with
pcDNA3); lane 2, level of expression in the absence of
GA. Lanes 3-7 show the level of Go
expression after exposure to 2, 20, 200, 2000, and 20,000 nM GA respectively. The Go band is marked by
the closed arrows, and the background band in COS cells is
marked by the open arrow. The autoradiograms were scanned by
a Hewlett Packard ScanJet 4c into Adobe Photoshop, and the density of
bands was determined using NIH Image 1.61 (Wayne Rasband, National
Institutes of Health, Bethesda, MD). The level of Go was
set to 100%, and the relative density of Go bands is
shown below. Similar results were obtained in three independent
experiments.
o in the
presence of GA could arise from effects on the rate of
Go synthesis and/or degradation. To distinguish among
these possibilities, the rate of Go synthesis in PC12
and COS cells was determined by comparing the amount of
[35S]methionine/[35S]cysteine incorporated
into Go at various times after labeling, and the
Go degradation rate was measured by pulse-chase
experiments. Fig. 2A shows
that the amount of label incorporated at 30, 60, 90, and 120 min was
indistinguishable in PC12 cells in the presence or absence of GA.
Similar results were obtained in transiently transfected COS cells (not
shown). The rate of degradation was significantly faster in the
presence of GA for endogenous Go in PC12 cells and in
transfected COS cells (Fig. 2B). Not surprisingly, the
half-life of endogenous Go in PC12 cells is
significantly longer (>24 h) than the half-life of Go
transiently expressed in COS cells (~6 h). Nevertheless, the effect
of GA was similar in the two systems: a significant increase in the
amount of degradation was evident at nearly every time point. Taken
together, these results indicate that the predominant effect of GA on
steady state Go levels is through accelerated
degradation.
View larger version (28K):
[in a new window]
Fig. 2.
GA effects on rates of synthesis and
degradation of G o.
A, rate of synthesis. Confluent PC12 cells were pretreated
with or without GA (2 µM) for 24 h and exposed to a
Tran35S-label pulse (200 µCi/ml of
methionine/cysteine-free medium) at time 0. At 30, 60, 90, and 120 min
after labeling, cells were scraped and lysed in Buffer B, and 400 µg
of total protein was immunoprecipitated with R4 anti-Go
antibody in Buffer B, separated on 13% SDS-PAGE, and visualized by
autoradiography. A representative autoradiogram is shown in the
inset, and mean densitometry units (± the range) are
plotted over time. 
, absence of GA; 
, presence of GA.
B, rate of degradation. PC12 cells or
Go-transfected COS cells (analyzed at 48 h) were
cultured in the absence and presence of GA (2 µM, 18 h COS, 24 h PC12) and then incubated in methionine/cysteine-free
medium for 1 h followed by labeling with Tran35S-label
methionine/cysteine-free medium for 60-90 min. Monolayers were then
chased with unlabeled methionine/cysteine-containing medium for
different periods of time. Immunoprecipitations were done as described
under "Experimental Procedures." Gels were exposed for 12-48 h,
and bands were analyzed as described in Fig. 1. For PC12 cells, the
mean of three independent experiments (± S.E.) is plotted over time,
and for COS cells a representative experiment (n = 2)
is shown. For both cell lines, immunoprecipitated Go is
shown below.
Most intracellular protein degradation is catalyzed by calpains,
lysosomal proteases, or by the ubiquitin-proteosome system (reviewed in
Ref. 20). To test which of these pathways was responsible for the
accelerated degradation of Go, a series of well
characterized peptide aldehyde protease inhibitors were utilized (21).
Two of these peptides, Z-LLL and N-Ac-LLM, exhibit similar
inhibitory activity against calpains and lysosomal cathepsins, but
Z-LLL is a more potent proteosome inhibitor than N-Ac-LLM
(21, 22). Fig. 3 shows that when COS
cells expressing Go are treated with GA plus an
inhibitor of proteolysis (lanes 2-4) there are different effects on the levels of Go. In comparison with no
inhibitor, treatment with N-Ac-LLM has no significant effect
on Go levels (lane 3), and this is consistent
with little degradation occurring through the calpain and lysosomal
pathways. Z-LLL also blocks the proteosome pathway, and this inhibitor
was partially capable of blocking degradation of Go
induced by GA (lane 2). This finding suggests that
degradation of Go occurs through the proteosome pathway.
To confirm this, we used lactacystin (LCN), the most specific inhibitor
of the proteosome (inhibits all five proteolytic activities) (23, 24).
As is seen in Fig. 3, lane 4, lactacystin significantly
blocks degradation of Go (closed arrow). When
the Western blots of total cellular extracts are exposed for increasing lengths of time, higher molecular mass species gradually become apparent (Fig. 3, bottom, lanes 2-4). The
open arrows highlight the appearance of new bands initially
between Go and the 50-kDa background band (lanes
2 and 3), whereas longer exposures show the development
of new bands in the 50-110-kDa range (lane 4). Vector-transfected cells (lane 1) were maximally exposed to
indicate the background bands. This pattern of higher molecular mass
bands in the presence of LCN is characteristic of polyubiquitination, a
covalent modification that targets proteins for degradation by the
proteosome (reviewed in Ref. 20). This finding is consistent with
degradation of Go through polyubiquitination and is
similar to the yeast G subunit Gpa1, which has been shown to be
degraded by this pathway (6).
|
The effects of GA on Go degradation are highly
suggestive of an interaction between Go and hsp90.
However, hsp90 complexes in cells are often of low affinity and
disrupted by the detergents required for immunoprecipitations, and we
were unable to detect this interaction in COS cells. As an alternative
approach, we utilized the rabbit reticulocyte lysate system to look for
this interaction. Rabbit reticulocyte lysate is rich in hsp90 and has been used to study the folding of several molecules (7, 9). Fig.
4 shows that
[35S]methionine-labeled Go can be detected
in a complex with hsp90 although other control proteins are not
detected (not shown). The control immunoprecipitations with nonspecific
antibody contain almost no background (Fig. 4). Go was
consistently detected in hsp90 immunoprecipitates (n = 4), but the fraction precipitated was low (<10% of starting
material). The requirements for detergent and the low-affinity nature
of the interaction between hsp90 and Go are the most
likely explanations for this result. The hsp90 antibody can precipitate
hsp90 alone or in a complex with other proteins, so we cannot exclude
the possibility that the interaction of Go with hsp90 is
indirect. The N terminus of Go is important for
interactions with G protein G subunits and for interactions with
the plasma membrane (12, 13, 18). Myristoylation of the N-terminal
glycine (first amino acid after cleavage of methionine) and
palmitoylation on cysteine 2 are also important to these functions. However, mutation of N-terminal glycine to alanine (G1A) did not disturb the interaction with hsp90 (Fig. 4), and likewise, this mutation in Gpa1 did not affect its degradation through the proteosome pathway (6). Similar results were obtained with other Go
mutants previously characterized (13, 18), which are deleted up to 20 amino acids from the N terminus (not shown). These results indicate
that myristoylation and an N-terminal amino acid sequence are not
necessary for association of Go with the hsp90
complex.
|
The identity of signaling molecules and their levels expressed in the
membrane play an important role in signal transduction specificity.
Critical to determining these levels are the mechanisms through which G
protein subunits are degraded. The results described above, and
studies in yeast (6), are consistent with degradation of G subunits
through the ubiquitin-proteosome pathway. Several studies have
demonstrated that activated G subunits (through cholera toxin
(Gs), by activating mutation, or by receptor
stimulation) are more rapidly degraded, but the mechanisms of
degradation are not addressed (25-27). Furthermore, G subunits have
different half-lives depending upon the cell type. In GH4 cells the
half-life of Go is about 28 h, but is greater than
72 h in cardiac myocytes (28). These different half-lives occur,
in part, because of differences in rates of protein degradation (28).
Our results suggest that steady state Go protein levels
are regulated by interaction with an hsp90 complex that prevents
degradation through the proteosome pathway and that N-terminal
myristoylation is not required for this interaction. Although the time
course and dose responses of GA treatment will vary among cell lines,
the observation that this mechanism appears to be preserved in a
transient expression system will make more detailed study of these
mechanisms feasible. Other cytosolic proteins are likely to participate
in this process and together provide an important mechanism for
regulating G levels and function.
| |
ACKNOWLEDGEMENT |
|---|
We thank Dr. Eva Neer for continued support of this work.
| |
FOOTNOTES |
|---|
* This work was supported by the National Institutes of Health and by a Massachusetts Affiliate American Heart Association Beginning Grant-in-aid (both to B. M. D.).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.
Career Investigator of the Consejo Nacional de Investigaciones
Cientìficas y Tecnológicas, Argentina.
¶ To whom correspondence should be addressed: FIBA (Fundación para Investigaciones Biológicas Aplicadas)-INBIOP, Vieytes 3103, 7600 Mar del Plata, Argentina. Tel.: 54-223-474-8784; Fax: 54-223-475-7120; E-mail: lbusconi@hotmail.com.
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ABBREVIATIONS |
|---|
The abbreviations used are: G protein, guanine nucleotide-binding protein; hsp90, 90-kDa heat shock protein; GA, geldanamycin; PAGE, polyacrylamide gel electrophoresis; Z-LLL, benzyloxycarbonyl-leucinyl-leucinyl-leucinyl-H; LCN, lactacystin; N-Ac-LLM, N-acetyl-leucinyl-leucinyl-methional-H; HA, hemagglutinin.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Y. Wang and H. G. Dohlman Regulation of G Protein and Mitogen-Activated Protein Kinase Signaling by Ubiquitination: Insights From Model Organisms Circ. Res., December 8, 2006; 99(12): 1305 - 1314. [Abstract] [Full Text] [PDF]
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 L. Mouledous, J. Neasta, S. Uttenweiler-Joseph, A. Stella, M. Matondo, M. Corbani, B. Monsarrat, and J.-C. Meunier Long-Term Morphine Treatment Enhances Proteasome-Dependent Degradation of G{beta} in Human Neuroblastoma SH-SY5Y Cells: Correlation with Onset of Adenylate Cyclase Sensitization Mol. Pharmacol., August 1, 2005; 68(2): 467 - 476. [Abstract] [Full Text] [PDF]
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 D. Zhu, K. S. Kosik, T. E. Meigs, V. Yanamadala, and B. M. Denker G{alpha}12 Directly Interacts with PP2A: EVIDENCE FOR G{alpha}12-STIMULATED PP2A PHOSPHATASE ACTIVITY AND DEPHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN, Tau J. Biol. Chem., December 31, 2004; 279(53): 54983 - 54986. [Abstract] [Full Text] [PDF]
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J. Luo and J. L. Benovic G Protein-coupled Receptor Kinase Interaction with Hsp90 Mediates Kinase Maturation J. Biol. Chem., December 19, 2003; 278(51): 50908 - 50914. [Abstract] [Full Text] [PDF]
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 T. Fischer, L. De Vries, T. Meerloo, and M. G. Farquhar Promotion of G{alpha}i3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP PNAS, July 8, 2003; 100(14): 8270 - 8275. [Abstract] [Full Text] [PDF]
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 R. Sathiyaa and M. M. Vijayan Autoregulation of glucocorticoid receptor by cortisol in rainbow trout hepatocytes Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1508 - C1515. [Abstract] [Full Text] [PDF]
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 W. B. Pratt and D. O. Toft Regulation of Signaling Protein Function and Trafficking by the hsp90/hsp70-Based Chaperone Machinery Experimental Biology and Medicine, February 1, 2003; 228(2): 111 - 133. [Abstract] [Full Text] [PDF]
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A. A. Waheed and T. L. Z. Jones Hsp90 Interactions and Acylation Target the G Protein Galpha 12 but Not Galpha 13 to Lipid Rafts J. Biol. Chem., August 30, 2002; 277(36): 32409 - 32412. [Abstract] [Full Text] [PDF]
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 B. Junoy, H. Maccario, J.-L. Mas, A. Enjalbert, and S. V. Drouva Proteasome Implication in Phorbol Ester- and GnRH-Induced Selective Down-Regulation of PKC ({alpha}, {epsilon}, {zeta}) in {alpha}T3-1 and L{beta}T2 Gonadotrope Cell Lines Endocrinology, April 1, 2002; 143(4): 1386 - 1403. [Abstract] [Full Text] [PDF]
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R. Vaiskunaite, T. Kozasa, and T. A. Voyno-Yasenetskaya Interaction between the Galpha Subunit of Heterotrimeric G12 Protein and Hsp90 Is Required for Galpha 12 Signaling J. Biol. Chem., November 30, 2001; 276(49): 46088 - 46093. [Abstract] [Full Text] [PDF]
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R. Kumar, N. Grammatikakis, and M. Chinkers Regulation of the Atrial Natriuretic Peptide Receptor by Heat Shock Protein 90 Complexes J. Biol. Chem., March 30, 2001; 276(14): 11371 - 11375. [Abstract] [Full Text] [PDF]
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