Figure 3. Genetic and Biochemical Analysis of the VAB-1 Eph/MSP Receptor Pathway(A-C) MSP binds the VAB-1 ectodomain (VAB-1ECT).(A) VAB-1ECT with its endogenous signal peptide and V5 and 6-His epitope tags was expressed in 293F cells.(B) Western blot probed with V5 antibodies showing VAB-1ECT secretion into the culture medium.(C) In vitro interaction between MSP and VAB-1ECT. Purified MSP-142 (100 nM) was incubated with partially purified VAB-1ECT (14 nM), and the complex was isolated by immunopreciptation with MSP antibodies and protein-A Sepharose (lanes 1-4) or by Ni-NTA agarose (lanes 5-7).(D-K) Evidence that PQN-19, PKC-1, DAB-1, and VAV-1 function with the VAB-1 Eph/MSP receptor.(D) Organization of signaling domains in VAB-1 pathway proteins. PQN-19 contains VHS (VPS-27/Hrs/STAM), UIM (ubiquitin-interaction motif), and SH3 domains. PKC-1 contains C1, C2, kinase, and protein kinase C domains. DAB-1 contains a phosphotyrosine binding (PTB) domain. VAV-1 contains CH (calponin homology), C1, RhoGEF, SH2, and SH3 domains.(E) Measurement of oocyte meiotic maturation rates after
dab-1,
vav-1,
pqn-19,
pkc-1, or control RNAi in
fog-2(
q71),
vab-1(
dx31);
fog-2(
q71), or
ceh-18(
mg57);
fog-2(
q71) female genetic backgrounds. Each of the four genes synergize with
ceh-18 but not
vab-1. Error bars represent SD.(F-K) Fluorescence micrographs showing MAPK-YT staining (red) in oocytes.(F) In wild-type hermaphrodites, MSP-dependent MAPK-YT staining is observed in proximal oocytes (typically oocytes -1 through -3).(G) MAPK-YT staining is extended to three to eight proximal oocytes in
vab-1(null) hermaphrodites, consistent with the idea that
vab-1 is a negative regulator of meiotic maturation and MAPK activation in oocytes [7].(H-K) Similarly,
dab-1(null) (H),
pqn-19(null) (I),
pkc-1(null) (J), and
vav-1(RNAi) hermaphrodites (K) exhibit an extended MAPK-YT staining pattern similar to that of
vab-1(null) hermaphrodites (G).(L-O) DAB-1 is expressed in oocytes. Western blot detection of DAB-1 (53 kDa) in the wild-type, but not
dab-1(
gk291) (L), hermaphrodites. Fluorescent detection of DAB-1 in oocytes from wild-type hermaphrodites (M) and
fog-2(
q71) females (N), but not
dab-1(
gk291) hermaphrodites (O). DAB-1 is cortically enriched when sperm are present.