ACCELERATED PUBLICATION
Ubiquitylation of MEKK1 Inhibits Its Phosphorylation of MKK1 and
MKK4 and Activation of the ERK1/2 and JNK Pathways*
James A.
Witowsky and
Gary L.
Johnson
From the Department of Pharmacology, University of Colorado Health
Sciences Center and University of Colorado Cancer Center,
Denver, Colorado 80262
Received for publication, November 4, 2002, and in revised form, November 25, 2002
 |
ABSTRACT |
MEKK1 is a MAPK kinase kinase that is activated
in response to stimuli that alter the cytoskeleton and cell shape.
MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and
JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been
shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945-956).
MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1
ubiquitylation is inhibited by mutation of cysteine 441 to alanine
(C441A) within the PHD. The functional consequence of MEKK1
ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of
MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and
preserves the ability of MEKK1 to catalyze MKK1 and MKK4
phosphorylation. MEKK1 ubiquitylation represents a mechanism for
inhibiting the ability of a protein kinase to phosphorylate substrates
and regulate downstream signaling pathways.
 |
INTRODUCTION |
Ubiquitin is a highly conserved 76-amino acid globular protein
that was identified as the first protein to act as a covalent modifier.
Ubiquitylation is the process of attachment of ubiquitin to a target
protein and is a multistep process that involves the action of at least
three classes of enzyme: the ubiquitin-activating enzyme
(E1),1 a
ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), which
assists in substrate recognition and the transfer of ubiquitin to the
target protein. There are two main types of E3 enzyme, those that
contain a homology to E6 C
terminus (HECT) domain and those that have a
really interesting new
gene (RING finger or RING finger-like) domain
(1). The plant homeobox domain
(PHD) is a RING finger-like domain defined by a series of cysteine and histidine residues with a characteristic spacing that mediates coordination of two zinc ions in a cross-brace structure (2). Disruption of a metal coordinating residue within the RING finger or
PHD has been shown to inactivate the ligase properties of the enzyme
(3).
Classically, ubiquitin is involved in the homeostasis of
cellular proteins by removing unnecessary, deleterious, or misfolded proteins primarily through the ubiquitin-proteasome degradation pathway
(4). Ubiquitin has been shown to function in many cellular processes
including cell cycle progression, apoptosis, cell differentiation, and
DNA repair (5). Recently, ubiquitylation of specific proteins has been
shown to have functions independent of proteasome-mediated degradation
(6). For example, vesicular sorting and TRAF6 organization of the TAK1
signal transduction complex has been shown to involve ubiquitylation of
proteins for the control of protein-protein interactions (7-9).
MEKK1 is a 196-kDa serine/threonine MAPK kinase kinase that can
regulate both the extracellular-regulated kinase (ERK1/2) and the c-Jun
NH2-terminal kinase (JNK) pathways in response to specific
stimuli (10). We and others have shown that MEKK1 is activated by a
variety of stimuli that alter cell shape and is required for normal
cell motility (11, 12). MEKK1 is a complex protein containing multiple
identified domains that may serve to regulate function. The E3 activity
of the PHD of MEKK1 was recently described by Lu et al.
(13). Here we demonstrate that full-length MEKK1 requires an intact PHD
for its own ubiquitylation, and this ubiquitylation impairs its ability
to phosphorylate MKK1 and MKK4.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction--
The cysteine to alanine mutation at
position 441 of MEKK1 was made in pCMV5 using a PCR strategy. The 5'
oligonucleotide CAG ATG TGT CCG ATC GCC TTG CTG GGC that encodes the
mutation and a PvuI restriction site was used in conjunction
with the 3' oligonucleotide TAC CTA GCA TGA ACA GAT TGG GCC CCA CTA ACG
TCG TGG G. The 3' primer binds to a region of MEKK1 including the
ApaI site at position 1769 followed by a stretch of
non-complimentary sequence. The 469-bp product was purified and
extended in a second reaction using the MEKK1pCMV5 vector as template
for 5 cycles. The 5' primer CAC AAA AGC TTG CCA CCA TGG ATC TGT ACG ATG
ACG ATA AGG CGG CGG adds a HindIII site and a Xpress epitope
tag to MEKK1. The product was purified from an agarose gel, digested
with HindIII and ApaI, and ligated into the
pCMV5-HA-MEKK1 construct that had been cleaved with the same enzymes.
Colonies were screened with PvuI and sequence-verified.
Purification of Ubiquitylated Proteins--
Lysates expressing
the indicated proteins were prepared, and the protein concentration was
determined by the Bradford method. 0.5-1.0 mg of total protein was
brought to a volume of 500 µl with lysis buffer and rotated
end-over-end at 4 °C with 30 µl of Ni-NTA beads
(PharMingen). After the 2-4-h incubation, the beads were washed
three times with lysis buffer, and the beads were eluted in 100 µl of
150 mM imidazole.
MEKK1 Kinase Assays--
500 µg of total protein
from lysates was immunoprecipitated with antibodies to the
NH2-terminal epitope tag of the expressed proteins.
Complexes were washed twice in lysis buffer and once in kinase buffer
(20 mM HEPES, pH 7.5, 10 mM MgCl2,
5 mM p-nitrophenyl phosphate). The beads
were then incubated for 20 min at 30 °C in 50 µl of kinase buffer
supplemented with 10 µCi of [
-32P]ATP and 1 µg of
either GST-MKK1 or GST-MKK4 (Upstate Biotechnology Inc.). Endogenous
JNK activity was determined as described previously (14).
 |
RESULTS |
Expression of MEKK1 Stimulates Ubiquitylation--
Within the
MEKK1 protein sequence, multiple protein binding regions and domains
have been identified that may regulate its cellular localization and
activity (Fig. 1A). The
COOH-terminal region contains the kinase domain, two ubiquitin
interaction motifs (UIMs), a region of interaction with small GTPases,
and a caspase 3-like cleavage site. Cleavage of MEKK1 by caspases
generates a catalytically active 91-kDa COOH-terminal fragment and a
105-kDa NH2-terminal fragment containing the zinc
finger-like PHD. To determine whether MEKK1 stimulates ubiquitylation
in vivo, we expressed HA-tagged wild type and
kinase-inactive MEKK1 in cells in the presence or absence of
His6-Myc-tagged ubiquitin (Fig. 1B). Cell
lysates were separated on SDS-PAGE and Western blotted with the
anti-Myc antibody 9E10 to recognize ubiquitin-conjugated proteins. A
dramatic increase in immunoreactivity is seen in the lysates
co-expressed with wild type MEKK1 and ubiquitin. The smear of
immunoreactivity seen is characteristic of ubiquitin modifications of
multiple proteins in the cell lysate. Therefore, MEKK1 stimulates covalent modification of proteins with ubiquitin (Fig. 1B).
The stimulation is a specific function of MEKK1, and Myc
immunoreactivity is not seen in the control lysate from cells
transfected with empty vector with ubiquitin. The kinase activity of
MEKK1 is required for the dramatic increase in protein ubiquitylation.
Expression of kinase-inactive MEKK1 shows only a modest increase of Myc
immunoreactivity over the background. Quantitation of the
autoradiograph indicates that ubiquitylation stimulated by
kinase-inactive MEKK1 is less than 10% of the ubiquitylation seen with
wild type MEKK1. Consistent with our findings, the PHD of MEKK1 was
recently reported to possess a ubiquitin ligase activity that was more
active in wild type than in kinase inactive MEKK1 (13).

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Fig. 1.
MEKK1 stimulates ubiquitylation in a
kinase-dependant manner and requires an intact full-length
protein. A, the relative positions of known protein
binding domains, truncations, and point mutations of MEKK1 are
indicated. HEK293 cells were transfected with either empty vector
(EV), MEKK1, or a kinase-inactive mutant of MEKK1
(MEKK1K ). B, cells were treated
with 20 µM proteasome inhibitor, MG-132, and the cell
lysates were separated on 10% SDS-PAGE and Western-blotted with 9E10,
a monoclonal anti-Myc antibody. C, in addition to empty
vector (EV) and MEKK1, the NH2-terminal 719 amino acids (1-719) and the 91-kDa COOH-terminal fragment
were expressed separately or together in the presence or absence of
His6-Myc-ubiquitin and probed as in B. Ub, ubiquitin.
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Our laboratory has shown expression of the 91-kDa COOH-terminal kinase
fragment of MEKK1 activates ERK1/2 and JNK pathways (15). We sought to
determine whether ubiquitin ligase activity observed with MEKK1
expression required a functional kinase domain within the full-length
MEKK1 protein. To test this, we compared the full-length MEKK1 protein
to expression of the PHD containing amino-terminal fragment (1-719
amino acids) and the 91-kDa kinase-active carboxyl-terminal fragment
alone or in combination in the presence or absence of
His6-Myc-ubiquitin (Fig. 1C). Total cell lysates were Western blotted with anti-Myc antibody. An intense smear of
immunoreactivity is observed with full-length kinase-active MEKK1 in
the presence of ubiquitin. Overexposure of the nitrocellulose membrane
reveals that expression of the NH2-terminal
fragment behaves similar to the kinase-inactive full-length protein, as there is only a modest increase of immunoreactivity over the control lysates. Expression of the COOH-terminal kinase fragment alone or in
combination with the PHD-containing NH2 terminus stimulates ubiquitylation at a modest but higher level than observed with the
NH2 terminus alone (Fig. 1C). Co-expression of
the NH2- and COOH-terminal regions of MEKK1 did not enhance
protein ubiquitylation above that observed with the kinase domain
alone. These experiments demonstrate a requirement for full-length
kinase-active MEKK1 for significant stimulation of ubiquitylation.
MEKK1 Ubiquitylation Requires a Functional PHD--
To determine
whether a functional PHD is required for the ubiquitylation of MEKK1 in
the full-length protein, we mutated cysteine 441 to alanine. Cysteine
441 is predicted to be a critical zinc-coordinating residue in the RING
finger-related PHD based on similar mutation made in the RING finger of
BRAC1 (3). Wild type and C441A MEKK1 were transfected in the presence
or absence of His6-Myc-ubiquitin. The cell lysates were
Western blotted for expression of the proteins with the anti-MEKK1
antibody, C22 (Fig. 2A). In
the absence of ubiquitin, MEKK1 and C441A are expressed at similar
levels. Co-expression of ubiquitin and wild type MEKK1 results in the
ubiquitylation of full-length MEKK1 protein that migrates as larger
molecular weight species. C441A is not significantly ubiquitylated, and slower migrating bands are not observed relative to C441A MEKK1 expressed in the absence of ubiquitin. The 91-kDa COOH-terminal fragment is generated from both wild type and C441A MEKK1 proteins. In
the presence of ubiquitin, the 91-kDa COOH-terminal fragment derived by
proteolytic cleavage of wild type MEKK1 (16) forms a ladder in ~8-kDa
increments consistent with the fragments being multiubiquitylated. The
COOH-terminal fragment generated from C441A MEKK1 is largely
distributed among three bands of reactivity with the most prominent at
91 kDa and represents a non-ubiquitylated MEKK1 kinase domain. A strong
band is seen at ~100 kDa and a weak band at 108 kDa, which
predictably represent a mono- and diubiquitylated COOH-terminal
fragments, respectively. Affinity precipitation of the lysates with
NTA-Ni beads followed by immunodetection of MEKK1 confirmed that
full-length MEKK1, but not the C441A MEKK1 PHD mutant, is ubiquitylated
(Fig. 2B). The amount of COOH-terminal fragment eluted from
the Ni2+ beads may simply be contamination in the bead pull
down or due to the presence of two overlapping UIMs. The UIMs could
possibly associate with ubiquitylated proteins including MEKK1 in the
lysate. A band of ~100 kDa in the wild type MEKK1 lane correlates
with the 91-kDa MEKK1 fragment that is monoubiquitylated with
endogenous ubiquitin, suggesting that C441A MEKK1, possibly through the
UIM, has a limited ability to be ubiquitylated.

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Fig. 2.
Mutation of the PHD domain inhibits MEKK1
ubiquitylation. A, empty vector (EV), wild
type MEKK1, or C441A mutant of MEKK1 was transiently expressed in the
presence or absence of His-Myc-ubiquitin (Ub) in HEK293
cells. The cells were treated as described previously. 100 µg of
protein in cellular lysate was separated on SDS-PAGE and
Western-blotted with the anti-MEKK1 antibody, C22, or 1000 µg of
lysate was incubated with 30 µl Ni-NTA beads for 4 h, washed,
eluted in 150 mM imidazole and Western-blotted with C22
(B).
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MEKK1 Ubiquitylation Inhibits in Vitro Substrate
Phosphorylation--
To determine whether ubiquitylation modulated
MEKK1 kinase activity, we performed in vitro kinase assays
of MEKK1 isolated from cells using MKK1 and MKK4 as substrates (Fig.
3). A ladder of ubiquitylated MEKK1 is
seen in the presence of co-transfected ubiquitin. Strikingly,
ubiquitylation of MEKK1 leads to a significant inhibition of substrate
phosphorylation. In contrast, MKK1 and MKK4 phosphorylation by C441A is
unaffected by expression of ubiquitin. These results indicate that
PHD-dependent ubiquitylation of MEKK1 inhibits the ability
of MEKK1 to phosphorylate MKK1 and MKK4 in vitro.

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Fig. 3.
MEKK1 in vitro kinase
activity toward MKK1 and MKK4 is inhibited by ubiquitylation. 500 µg of lysate from cells transfected with empty vector
(EV), MEKK1, or C441 MEKK1 were immunoprecipitated with
amino-terminal antibodies. The precipitated complexes were assayed for
kinase activity using 1 µg of GST-MKK1 (A) or GST-MKK4
(B) as substrate. A and B, lower
panels, reactions were transferred to a nitrocellulose membrane,
and autoradiography was employed to visualize incorporation of
32P into substrate. A and B,
upper panels, total MEKK1 in the reaction was detected with
the anti-MEKK1 antibody, C22. Ub, ubiquitin.
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MEKK1 Ubiquitylation Inhibits ERK1/2 and JNK
Activation in Cells--
The ability of MEKK1 and C441A MEKK1 to
activate ERK1/2 and JNK pathways in cells was investigated (Fig.
4). Activation of the ERK1/2 MAPK pathway
was assessed by a phosphospecific antibody to ERK1/2 that specifically
recognizes phosphorylation of the activating residues in the T-loop of
the kinase (Fig. 4B). An antibody that cross-reacts with
both ERK1 and ERK2 was used to detect total ERK1/2 protein (Fig.
4C). Expression of MEKK1 (Fig. 4A) activated the
ERK1/2 pathway, and this stimulation was lost with the co-expression of
ubiquitin (Fig. 4B). Additionally, there is an apparent
increase in the total amount of MEKK1 protein with ubiquitin
co-expression. The C441A mutant in the presence or absence of ubiquitin
activated the ERK1/2 pathway to a greater extent than wild type MEKK1.
These results are consistent with ubiquitylation of MEKK1 being a
negative regulator of MEKK1 activation of the ERK1/2 pathway.

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Fig. 4.
Inhibition of MEKK1-stimulated ERK1/2 and JNK
pathways by ubiquitylation in vivo. A
and D, HEK293 cells were transfected with the indicated
constructs, and clarified total cell lysates were Western-blotted for
MEKK1 using C22. B, phosphorylated ERK1 and ERK2 were
detected using a phospho-specific antibody for p-ERK1/2. C,
the blot was stripped and reprobed for total ERK1 and ERK2 using a
cross-reactive ERK1/2 antibody. E, JNK kinase activities
were performed by affinity precipitation with GST-c-Jun 1-79 as
described previously. F, total JNK was immunodetected with
an anti-JNK antibody. Ub, ubiquitin.
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A similar inhibitory effect of MEKK1 ubiquitylation was observed for
the JNK pathway. Cells were treated as described previously, and
lysates were prepared. JNK activity was measured by affinity precipitation of activated JNK with GST-c-Jun (amino acids 1-79) (Fig.
4E). Total JNK and MEKK1 were visualized by immunodetection with their respective antibodies shown in Fig. 4, D and
F. To achieve comparable protein expression levels for the
wild type MEKK1 protein in the presence and absence of ubiquitin, the
total amount of DNA transfected was reduced to 100 ng per dish. This low amount of DNA results in a somewhat modest JNK activation with the
wild type MEKK1 that is significantly reduced with co-expression of
ubiquitin. The activity of C441A MEKK1 is maintained in the presence of
expressed ubiquitin compared with C441A alone.
Ubiquitylated MEKK1 Is Expressed in the Absence of
Proteasome Inhibitor--
Because ubiquitylation of proteins is often
a signal for degradation by the proteasome, we determined whether
MEKK1-stimulated ubiquitylation of MEKK1 caused the loss of MEKK1
protein in cells. MEKK1 or C441A MEKK1 was expressed in the presence or
absence of His6-Myc-ubiquitin. The cells were treated with
Me2SO or the protease inhibitor MG-132, and the
clarified lysates were separated by SDS-PAGE. Immunodetection of MEKK1,
ERK1/2, and JNK revealed little difference between cells treated with
MG-132 or vehicle (Fig.
5A).

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Fig. 5.
Ubiquitin (Ub) modification
of MEKK1 does not target MEKK1 for rapid proteasome degradation.
Empty vector (EV), MEKK1, or C441A was transiently expressed
in the presence or absence of His-Myc-ubiquitin in HEK293 cells and
either treated with Me2SO or 20 µM MG132 for
5 h. 100 µg of cell lysate was probed by Western blot with
either C22 (A) or anti-ERK2 (B).
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 |
DISCUSSION |
MEKK1 is unique among MAPK kinase kinases in that it encodes a PHD
in its NH2 terminus. The MEKK1 PHD has E3 ligase activity (13) that is dependent on the kinase activity of the full-length 196-kDa MEKK1 protein. Lu et al. (13) demonstrated that a
GST fusion of the MEKK1 PHD had E3 ligase activity in vitro.
Our work demonstrates that a functional PHD is required for MEKK1
poly/multiubiquitylation. Thus, activation of MEKK1 not only stimulates
its ability to phosphorylate and activate MKK1 and MKK4 in the ERK1/2
and JNK pathways but also leads to the ubiquitylation of MEKK1 itself.
This ubiquitylation consequently inhibits MEKK1-catalyzed
phosphorylation of MKK1 and MKK4, thereby down-regulating the MEKK1
activation of the ERK1/2 and JNK pathways. The inhibition of MEKK1
phosphorylation activity is independent of
proteasome-dependent degradation of MEKK1. In fact,
ubiquitylated MEKK1 seems to be a stable protein in cells. Although it
is difficult to quantitate because of the poly/multiubiquitylation
of MEKK1 and the consequent laddering in SDS-PAGE, it appears that
ubiquitylated MEKK1 actually accumulates in cells and is not degraded
significantly by the proteasome. We propose that the
ubiquitylation-dependent uncoupling of MEKK1-catalyzed phosphorylation of MKK1 and MKK4 represents a novel function for protein ubiquitylation.
It appears that the C441A MEKK1 protein has greater
activity toward MKK1 and MKK4 than wild type MEKK1, based on the
relative protein levels in the immunoprecipitations. This is consistent with the PHD having a negative regulatory function that correlates with
its E3 ligase activity. Certainly, other proteins that interact with
MEKK1 may be ubiquitylated by the PHD E3 ligase activity of MEKK1. The
E3 ligase function of MEKK1 is likely to down-regulate MEKK1 activation
of ERK1/2 and JNK and also ubiquitylate other proteins to regulate
their function and/or targeting to the proteasome for degradation.
How could MEKK1 ubiquitylation inhibit MKK1 and MKK4 phosphorylation?
The ubiquitin modification of MEKK1 may simply block binding of MKK1
and MKK4. Adjacent to the COOH-terminal kinase domain are two UIMs that
could interact with ubiquitins covalently bound to MEKK1 and induce a
steric hindrance for substrate interaction. The fact that a full-length
196-kDa MEKK1 is required for MEKK1 ubiquitylation suggests that an
intramolecular interaction is required for regulation of the MEKK1 E3
ligase activity. It should be noted that our studies were done using
transfection. We must still define the role of ubiquitylation of
endogenous MEKK1 in cells.
The rapidly expanding functions for ubiquitin modification of proteins
indicates that ubiquitin is used to regulate protein targeting,
scaffolding, and activity. In regards to positive regulation of signal
transduction, the polyubiquitylation of TRAF6 is involved in the
organization of p38 and NF
B signaling complexes (8, 9). In contrast,
activation of the E3 ligase activity of c-Cbl by Src kinase
phosphorylation of c-Cbl (17, 18) results in the ubiquitylation of Src
and the receptors for EGF (19-21) and PDGF (22). Thus,
c-Cbl down-regulates tyrosine kinase signaling. It has also been shown
in yeast that pheromone activation of the mating pathway involving the
MAPK, Fus3, stimulates a feedback ubiquitylation and degradation of
Ste11, a MAPK kinase kinase (23). Our results demonstrate that the PHD
of MEKK1 is required for inhibition of ERK1/2 and JNK activation by
MEKK1. The C441A MEKK1 mutant protein stimulates the ERK1/2 and JNK
pathways, and this activation is not down-regulated as is seen with
wild type MEKK1, showing the requirement of the PHD of MEKK1 for
ubiquitin-dependent inhibition of MEKK1 substrate
phosphorylation. The regulation of Ste11 and MEKK1, both MAPK kinase
kinases, by ubiquitylation appears different. Ste11 is degraded and
MEKK1 substrate phosphorylation is inhibited when each is modified with
ubiquitin. The function of the MEKK1 PHD is a previously unrecognized
mechanism for controlling the activity of a MAPK kinase kinase and the
turn-off of MAPK signaling in cells.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant DK37871.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.
To whom correspondence should be addressed: Dept. of Pharmacology,
Rm. 2809, SOM, University of Colorado Health Sciences Center, 4200 East
Ninth Ave., Denver, CO 80262. Fax: 303-315-1022; E-mail: gary.johnson@uchsc.edu.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.C200616200
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ABBREVIATIONS |
The abbreviations used are:
E1, ubiquitin-activating enzyme;
E2, ubiquitin-conjugating enzyme;
E3, ubiquitin ligase;
C441A, cysteine 441 to alanine mutation;
ERK, extracellular signal-regulated kinase;
GST, glutathione
S-transferase;
HA, hemagglutinin epitope tag;
HECT, homology to E6 COOH
terminus: His6, hexahistidine;
JNK, c-Jun
NH2-terminal kinase;
MAPK, mitogen-activated protein
kinase;
MEKK1, mitogen-activated protein kinase kinase kinase 1;
MKK1, mitogen-activated protein kinase kinase 1;
MKK4, mitogen-activated
protein kinase kinase 4;
PHD, plant
homeobox domain;
RING, really
interesting new gene;
UIM, ubiquitin interaction motif;
Ni-NTA, nickel-nitrilotriacetic
acid.
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REFERENCES |
1.
|
Glickman, M. H.,
and Ciechanover, A.
(2002)
Physiol. Rev.
82,
373-428[Abstract/Free Full Text]
|
2.
|
Pascual, J.,
Martinez-Yamout, M.,
Dyson, H. J.,
and Wright, P. E.
(2000)
J. Mol. Biol.
304,
723-729[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Hashizume, R.,
Fukuda, M.,
Maeda, I.,
Nishikawa, H.,
Oyake, D.,
Yabuki, Y.,
Ogata, H.,
and Ohta, T.
(2001)
J. Biol. Chem.
276,
14537-14540[Abstract/Free Full Text]
|
4.
|
Jennissen, H. P.
(1995)
Eur. J. Biochem.
231,
1-30[Abstract]
|
5.
|
Naujokat, C.,
and Hoffmann, S.
(2002)
Lab. Invest.
82,
965-980[Medline]
[Order article via Infotrieve]
|
6.
|
Marx, J.
(2002)
Science
297,
1792-1794[Free Full Text]
|
7.
|
Katzmann, D. J.,
Babst, M.,
and Emr, S. D.
(2001)
Cell
106,
145-155[Medline]
[Order article via Infotrieve]
|
8.
|
Deng, L.,
Wang, C.,
Spencer, E.,
Yang, L.,
Braun, A.,
You, J.,
Slaughter, C.,
Pickart, C.,
and Chen, Z. J.
(2000)
Cell
103,
351-361[Medline]
[Order article via Infotrieve]
|
9.
|
Wang, C.,
Deng, L.,
Hong, M.,
Akkaraju, G. R.,
Inoue, J.,
and Chen, Z. J.
(2001)
Nature
412,
346-351[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Yujiri, T.,
Sather, S.,
Fanger, G. R.,
and Johnson, G. L.
(1998)
Science
282,
1911-1914[Abstract/Free Full Text]
|
11.
|
Yujiri, T.,
Ware, M.,
Widmann, C.,
Oyer, R.,
Russell, D.,
Chan, E.,
Zaitsu, Y.,
Clarke, P.,
Tyler, K.,
Oka, Y.,
Fanger, G. R.,
Henson, P.,
and Johnson, G. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7272-7277[Abstract/Free Full Text]
|
12.
|
Xia, Y.,
Makris, C., Su, B., Li, E.,
Yang, J.,
Nemerow, G. R.,
and Karin, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5243-5248[Abstract/Free Full Text]
|
13.
|
Lu, Z., Xu, S.,
Joazeiro, C.,
Cobb, M. H.,
and Hunter, T.
(2002)
Mol. Cell
9,
945-956[Medline]
[Order article via Infotrieve]
|
14.
|
Heasley, L. E.,
Storey, B.,
Fanger, G. R.,
Butterfield, L.,
Zamarripa, J.,
Blumberg, D.,
and Maue, R. A.
(1996)
Mol. Cell. Biol.
16,
648-656[Abstract]
|
15.
|
Widmann, C.,
Gibson, S.,
and Johnson, G. L.
(1998)
J. Biol. Chem.
273,
7141-7147[Abstract/Free Full Text]
|
16.
|
Widmann, C.,
Gerwins, P.,
Johnson, N. L.,
Jarpe, M. B.,
and Johnson, G. L.
(1998)
Mol. Cell. Biol.
18,
2416-2429[Abstract/Free Full Text]
|
17.
|
Yokouchi, M.,
Kondo, T.,
Sanjay, A.,
Houghton, A.,
Yoshimura, A.,
Komiya, S.,
Zhang, H.,
and Baron, R.
(2001)
J. Biol. Chem.
276,
35185-35193[Abstract/Free Full Text]
|
18.
|
Kassenbrock, C. K.,
Hunter, S.,
Garl, P.,
Johnson, G. L.,
and Anderson, S. M.
(2002)
J. Biol. Chem.
277,
24967-24975[Abstract/Free Full Text]
|
19.
|
Levkowitz, G.,
Waterman, H.,
Zamir, E.,
Kam, Z.,
Oved, S.,
Langdon, W. Y.,
Beguinot, L.,
Geiger, B.,
and Yarden, Y.
(1998)
Genes Dev.
12,
3663-3674[Abstract/Free Full Text]
|
20.
|
Yokouchi, M.,
Kondo, T.,
Houghton, A.,
Bartkiewicz, M.,
Horne, W. C.,
Zhang, H.,
Yoshimura, A.,
and Baron, R.
(1999)
J. Biol. Chem.
274,
31707-31712[Abstract/Free Full Text]
|
21.
|
Levkowitz, G.,
Waterman, H.,
Ettenberg, S. A.,
Katz, M.,
Tsygankov, A. Y.,
Alroy, I.,
Lavi, S.,
Iwai, K.,
Reiss, Y.,
Ciechanover, A.,
Lipkowitz, S.,
and Yarden, Y.
(1999)
Mol. Cell
4,
1029-1040[Medline]
[Order article via Infotrieve]
|
22.
|
Rosenkranz, S.,
Ikuno, Y.,
Leong, F. L.,
Klinghoffer, R. A.,
Miyake, S.,
Band, H.,
and Kazlauskas, A.
(2000)
J. Biol. Chem.
275,
9620-9627[Abstract/Free Full Text]
|
23.
|
Esch, R. K.,
and Errede, B.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
9160-9165[Abstract/Free Full Text]
|
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