1. A Role for C. elegans Eph RTK Signaling in PTEN Regulation
Sarah Brisbin, Jun Liu, Jeff Boudreau, Jimmy Peng, Marie Evangelista, Ian Chin-Sang 
Developmental Cell 
Volume 17, Issue 4, Pages (October 2009)
DOI: /j.devcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
The VAB-1 Tyrosine Kinase Domain Physically Interacts with DAF-18/PTEN
(A) Schematic diagram of VAB-1 and the DAF-18/PTEN full-length protein and other regions tested for binding to VAB-1. The VAB-1 kinase region (669– 985aa shown by a dashed box) was used as bait in yeast two-hybrid assays and identified five independent clones of the daf-18/pten gene (last five shown). The full-length DAF-18 protein does not interact with VAB-1 in yeast two-hybrid assays. The interaction is strongest within the mid region of DAF-18 (476–803 aa). The C-terminal end, which has a predicted PDZ-binding motif (PDZbm) appeared to reduce the interaction with VAB-1; relative interaction strength is indicated by “+,” weak; “++,” medium; “+++,” strong lacZ activity.
(B) GST pull-down experiments confirm the interaction. The VAB-1 kinase domain fused to GST as well as the VAB-1 kinase-inactive G912E both show equal binding to MBP::DAF-18. Protein expression of GST and GST::VAB-1 is shown below by Coomassie staining.
(C) MBP::DAF-18 full-length copurifies with HIS-6::VAB-1 (intracellular). The DAF-18 (D137A) phosphatase-inactivating mutation increased binding to VAB-1. Levels of purified HIS-6::VAB-1 are shown below.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2 VAB-1 and DAF-18/PTEN Are Expressed in Similar Tissues
(A–M) VAB-1 and DAF-18 are expressed in the germline precursor cells (Z2/Z3), oocytes, and neuronal tissues. (A) VAB-1 expression in Z2/Z3 (∼comma-stage embryo). (B) DAF-18 expression in Z2/Z3 (∼100-cell stage). (C) VAB-1 and DAF-18 Z2/Z3 expression in L1 larvae. (D) VAB-1 is expressed in at least two of the proximal oocytes, whereas (E and G) DAF-18 is expressed in more than five oocytes in both wild-type and female animals. (F and H) Increased DAF-18 expression is observed in fem-1(hc17);vab-1(dx31) mutants, and nuclear expression is also apparent. (I) VAB-1 is expressed in a variety of neuronal tissues, whereas (J) DAF-18 expression appears to be restricted to several of the amphid neurons. (K–M) DAF-18 protein is not detectable in head neurons of wild-type (WT) animals, but it is present in vab-1(dx31) and in the daf-18 overexpressing line (quIs18).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3 VAB-1 Negatively Regulates DAF-18/PTEN at the Protein Level
(A) Western blot with DAF-18 antibodies detects endogenous DAF-18 protein in embryo lysates. Wild-type (WT) DAF-18 expression was standardized as relative intensity 1.0. The daf-18(ok480) appears to be a null allele, as no protein product is detected (relative intensity 0.34 ± 0.19). daf-18 genomic rescue (quIs18) (relative intensity 1.69 ± 0.31), vab-1(dx31) (relative intensity 2.87 ± 0.03), and vab-1(e2) (relative intensity 2.45 ± 0.81) show increased DAF-18 expression compared to wild-type.
(B) Increased VAB-1 signaling reduces DAF-18 expression. A quIs16(a Hs promoter myr::vab-1);quIs18(daf-18 genomic) double transgenic animal shows decreased DAF-18 expression (lowered by 15%) under heat-shock (HS) conditions when VAB-1 is constitutively active (left). DAF-18 expression is slightly affected (increased by 7%) by heat-shock conditions (right).
(C) daf-18(e1375) forms DAF-18 protein at 15°C (relative intensity 1.0), but not at 25°C. In a vab-1(dx31) background, daf-18(e1375) forms a DAF-18 protein product at both temperatures (relative intensities 1.54 ± 0.24 and 1.70 ± 0.17, respectively).
(D) The regulation of DAF-18 by VAB-1 is not at the transcript level. Wild-type and vab-1(dx31) have equal daf-18 transcript levels, as determined by RT-PCR (left). Total RNA level and 28 s and 18 s ribosomal subunits (right).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
Increased DAF-18/PTEN Expression Confers Increased Longevity and Sensitivity to Dauer Conditions
(A) vab-1(dx31);fem-1(hc17) and quIs18;fem-1(hc17) have a significantly longer life span than fem-1(hc17) (p < 0.001). vab-1(dx31);fem-1(hc17) and quIs18;fem-1(hc17) have mean life spans of ± 0.44 days and ± 0.45 days (n = 185 and 127, respectively), whereas fem-1(hc17) has a mean life span of ± 0.32 days (n = 137). Data are represented as mean ± Standard Error of the Mean (SEM).
(B) vab-1(dx31) and quIs18 formed significantly more dauer larva than wild-type on daf-2 RNAi (∗p < 0.01). A total of 14.01% ± 1.02% of vab-1(dx31) (n = 1577) and 45.0% ± 2.371% (n = 1102) of quIs18 formed dauer larva on daf-2 RNAi at 25°C compared to 4.95% ± 0.66% of wild-type (n = 1778).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5 DAF-18/PTEN Plays a Role in Oocyte Maturation with VAB-1
(A and B) daf-18(ok480) suppresses vab-1(dx31) oocyte defects. The increased number of unfertilized oocytes laid by vab-1(dx31) compared to vab-1(dx31);daf-18(ok480) after 48 hr of egg laying (green signifies a fertilized embryo; red signifies an unfertilized oocyte).
(C) Unfertilized oocytes are present among fertilized embryos in vab-1(dx31) worms (indicated by a white arrowhead).
(D) Over a 72 hr egg-laying period, vab-1(dx31) lays significantly more unfertilized oocytes than wild-type, daf-18(ok480), vab-1(dx31);daf-18(ok480), and age-1(m333) (∗p < 0.01) (n = 5976, 1426, 3224, 3645, and 4474, respectively). vab-1(dx31);daf-16(mu86) does not display significant suppression of the increased oocyte-laying phenotype (n = 2258).
(E) A day-by-day breakdown of laid objects reveals that vab-1(dx31) lays significantly more unfertilized oocytes than vab-1(dx31);daf-18(ok480) after 24 hr of egg laying (∗p < 0.05) (Day 1: vab-1(dx31), n = 2996; vab-1(dx31);daf-18(ok480), n = 1600; Day 2: vab-1(dx31), n = 2300; vab-1(dx31);daf-18(ok480), n = 1130; Day 3: vab-1(dx31), n = 1864; vab-1(dx31);daf-18(ok480), n = 915.
(F) The daf-18(ok480) mutation suppresses the increased ovulation rate (number of ovulations/hour) of vab-1(dx31) (∗p < 0.01). Data are represented as mean ± SEM (WT, n = 19; daf-18(ok480), n = 14; vab-1(dx31), n = 18; vab-1(dx31;daf-18(ok480), n = 24).
(G) daf-18(ok480) mutants can suppress vab-1(dx31)-activated MAPK expression. Activated MAPK (green) is expressed in upwards of 5 oocytes in vab-1(dx31) worms and in typically 1–3 oocytes of wild-type. In daf-18(ok480) and vab-1(dx31);daf-18(ok480) mutants, this expression is reduced to the two most proximal oocytes.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6
The VAB-1 Tyrosine Kinase and the DAF-18 Phosphatase Are Substrates for Each Other
(A) VAB-1 and DAF-18 MBP fusion proteins were coexpressed in E. coli. Tyrosine phosphorylation (PY) was assayed with the anti-phosphotyrosine (4G10) antibody on western blots. VAB-1 intracellular (581–1117 aa) was able to phosphorylate the binding fragment of DAF-18 (406–835 aa) (lane 1). VAB-1 also displays strong autophosphorylation (autophos.), and coexpression of full-length DAF-18 full-length reduces VAB-1autophosphorylation (lane 2). A kinase-inactive VAB-1 (G912E) does not phosphorylate DAF-18 (406–835 aa) (lane 3). A phosphatase-deficient DAF-18 (C169S) did not reduce VAB-1 autophosphorylation as much as wild-type DAF-18 (compare VAB-1 autophosphorylation levels, in lanes 2 and 4). The blot was stripped and reprobed to show relative quantities of VAB-1and DAF-18 proteins (below).
(B–G) DAF-18 has tyrosine phosphatase activity in vivo. (B) Schematic diagram of the C. elegans six touch neurons (only ALMs and PLMs are labeled), and boxed regions indicate the regions shown in the confocal projections below. (C) A constitutively active VAB-1 tyrosine kinase (MYR-VAB-1) expressed from the mec-4 promoter shows strong anti-phosphotyrosine (α-PY) staining (red) in the touch neurons. MYR-VAB-1 causes neuronal defects such as premature termination neuron defects (e.g., PLM truncated). (D) In wild-type animals, the touch neurons do not stain for PY, but endogenous PY can be detected in the gut epithelial cells (asterisk). (E) DAF-18 expressed in the touch neurons reduces MYR-VAB-1-induced PY (red). Arrows point to the position of one PLM and one ALM, showing the absence of PY in these neurons. DAF-18 expression in the touch neurons had no effect on the endogenous gut PY (asterisk). (F) The same worm in (E) stained for DAF-18 (green) showing strong DAF-18 expression in the touch neurons. Expression of DAF-18 could also partially rescue the premature termination defects (compare the length of PLM in [C] versus [F]). (G) A merged image of (E) and (F).
(H and I) In some neurons, overexpression of DAF-18 was not able to reduce MYR-VAB-1 PY levels, and, instead, the MYR-VAB-1-expressing neuron had the effect of reducing the DAF-18/PTEN protein level in that neuron. Arrow points to the right ALM, showing high MYR-VAB-1 activity (red) but low DAF-18 protein (green).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. Figure 7
Genetic Models of the DAF-18/VAB-1 Interaction for Regulation of Dauer/Life Span and Oocyte Maturation in C. elegans
(A) In neurons, VAB-1/EphR increases insulin signaling by inhibiting the action of DAF-18/PTEN on PIP3. In this manner, the AGE-1/PI3K kinase is not antagonized by DAF-18/PTEN activity, resulting in increased PIP3 and inhibition of the DAF-16/FOXO transcription factor responsible for the initiation of genes involved in life span and dauer formation.
(B) In oocytes, VAB-1/EphR receives a signal for oocyte maturation (MSP, major sperm proteins). In the absence of MSP, VAB-1/EphR acts to inhibit DAF-18/PTEN activity on MAPK. In the absence of VAB-1, DAF-18 signaling can positively regulate MAPK expression, resulting in increased oocyte maturation.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions