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The Jun N-terminal kinase kinase kinase MLK2 is required for the formation of the pronephros during early Xenopus development. Here, we have used a yeast 2-hybrid screen to identify proteins that interact with and regulate xMLK2. Using an N-terminal polypeptide encompassing the SH3 and kinase domains of xMLK2 as bait, five independent cDNAs were identified, all of which encoded a Xenopus ubiquitin conjugating enzyme, ube2d3.2. Ube2d3.2 is a functional E2 enzyme expressed maternally and in a tissue-restricted fashion during development. Ectopic expression of ube2d3.2 inhibits formation of the pronephric tubules, resulting in a phenotype very similar to the loss of xMLK2 function. Because ube2d3.2 is also shown to limit accumulation of xMLK2, it is likely that this effect is direct, although other explanations are possible. Ube2d3.2 is thus probably an endogenous regulator of xMLK2, and hence of JNK activity.
Fig. 2 Embryonic expression of ube2d3.2. (now called ube2d4) (A) A polyclonal antibody raised to amino acids 1–147 of ube2d3.2 was used to probe control embryos (uninjected) and embryos expressing FLAG-tagged ube2d3.2(-FLAG). (B) Equal quantities of total embryonic protein
from the indicated stages were loaded onto an SDS-PAGE gel and probed with the polyclonal antibody raised against ube2d3.2,
upper panel. A duplicate membrane was probed with pre-immune
serum from the rabbit used to produce the specific ube2d3.2 antibody, middle panel (both the upper and middle panels were exposed for the same time). Tubulin was used as a loading control lower panel. (
C) Expression pattern
of ube2d3.2 mRNA at stages
28 and 35. Panels show lateral
views with anterior to left and the
right panel is a magnification of
the middle panel. AS, antisense
probe. The somites (s), eye (e), otic
vesicle (o), branchial arches (ba),
and pronephric primordium (p)
Fig. 3 Ube2d3.2 is a functional UBC that regulates xMLK2 and
xMLK2 is ubiquitinated in vivo. (A) Xenopus laevis oocytes were
injected with untagged ube2d3.2 and FLAG-tagged ubiquitin.
Thirty-six hours after injection, groups of oocytes were treated with
MG-132 for 12 hr. Proteins were extracted and total protein from
20 oocytes was subjected to anti-FLAG (ubiquitin-specific)
immunoprecipitation. The resulting precipitate was loaded onto
an SDS-PAGE gel in comparison with the equivalent of total protein
from three oocytes (lysate) and probed with the anti-ube2d3.2
antibody. (B) xMLK2-HA was expressed in X. laevis embryos with
or without co-expression of ube2d3.2-FLAG. Embryos were allowed
to develop to stage 19 or 25 before total protein extracts were
prepared. Protein from the equivalent of three embryos was loaded
onto an SDS-PAGE gel and probed with an anti-HA antibody
(upper panel) and then reprobed with an anti-FLAG antibody
(lower panel). Numbers below the tracks indicate relative xMLK2
protein levels established from unsaturated blot exposures by peak
fitting. (C) xMLK2-FLAG was expressed in 293T cells together
with ube2d3.2 and with or without myc-tagged ubiquitin. The upper
panel shows ubiquitination of xMLK2 and ‘‘’’ indicates the
apparent molecular weight expected for mono-ubiquitination, while
the middle panel shows the amount of xMLK2 present in the IP.
The lower panels indicate the amount of xMLK2 and ube2d3.2 in
Fig. 4 Ube2d3.2 affects pronephros formation. (A) The left-hand
panel shows the dorsal view of a stage 45 embryo injected unilaterally
with ube2d3.2. The arrow on the right indicates the pronephros
and the arrow on the left labeled with an X indicates the
region normally occupied by the left-hand pronephros, which is
missing. The right-hand panels display higher magnifications; the
boxes indicate the regions shown enlarged in the lower two panels.
Fig. 4 Ube2d3.2 affects pronephros formation. (B) Stage 45 embryos were also longitudinally sectioned after unilateral
injection of ube2d3.2 mRNA at the two-cell stage. The gray
oval on the uninjected side indicates the pronephros and on the injected side the site normally occupied by the pronephros. For a complete range of longitudinal sections, see Supplementary
Fig. 4 Ube2d3.2 affects pronephros formation. (C) Two-cell stage embryos were injected unilaterally
with increasing amounts of ube2d3.2 or ube2d3.2-C85A mRNA
(0.5, 1, or 2 ng). Embryos were allowed to develop until stages
44/45 when the diameters of the pronephros and the eyes were
measured. Each measurement was normalized to the corresponding
organ diameter from the uninjected side of the same embryo.
The numbers above each bar represent the number of embryos
scored. (D) Embryos in (C) showing a greater than 20% reduction
in the pronephros diameter were scored as positive for a loss of
Fig. S1. Stage 45 embryos were longitudinally sectioned
after unilateral injection of ube2d3.2 mRNA at
the two-cell stage. A range of sections at given relative
levels through the pronephric tissues are shown. The
right-hand panels are shown at half the linear magni-
fication of the right-hand panels in order to present
sections spanning the complete width of the embryo.
Fig. 5 Pronephric markers 3g8 is affected
by ube2d3.2 injection and somites are not
affected. (A) Two-cell stage embryos were
injected unilaterally with 1.5ng of ube2d3.2
or ube2d3.2-C85A mRNA, and allowed to
develop until stage 37 before whole-mount
staining with the 3G8 antibody. Upper
panels show bright-field images of the injected
embryos, and the lower panels show
epifluorescence images of the region of the
pronephros. The three pronephric tubules
can be clearly distinguished in each case,
except where Ube2d3.2 is expressed.
(B) Two-cell stage embryos were injected
unilaterally with 750 pg of ube2d3.2
mRNA, and allowed to develop until stage
37 before whole-mount staining with the
12/101 antibody. The upper panels show
bright-field images of the injected embryos
and the lower panels show epifluorescence
images. Note that somite segmentation was
not affected by ube2d3.2 mRNA injection.
Fig. 6 Two-cell stage embryos were injected unilaterally with 1.5ng
of ube2d3.2 (A) or ube2d3.2C85A (B) mRNA, or with 1.5 pmol of
xMLK2 AS morpholino (C) (Poitras et al., 2003). Embryos were allowed to develop until stages 18/19, 21/24, and 29. They were then
processed for whole-mount in situ hybridization. The injected side in the
dorsal views (left panels) is oriented toward the bottom of the panels.
ube2d4 (ubiquitin conjugating enzyme E2D 4 (putative)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior left, dorsal up.