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Fig. 5. Drosophila Spry is phosphorylated and binds a
substrate-trapping form of Csw. (A) Tyrosine phosphorylation of
Spry. S2 cells expressing Breathless-FLAG were mock-treated (lanes 1, 3) or
treated with 0.1 mM pervanadate, a tyrosine phosphatase inhibitor (lanes 2, 4)
and endogenous Spry was immunoprecipitated from cell extracts and analyzed on
immunoblots. (Lanes 1, 2) Immunoblot probed with anti-phosphotyrosine to
detect phosphorylated Spry. (Lanes 3, 4) Immunoblot probed with anti-Spry to
detect all Spry (
70 kDa isoform, Spry70; doublet of 42 kDa isoforms,
Spry42). There is tyrosine phosphorylation of Spry70 and a Spry42 isoform
(lane 2). Similar results were obtained in three experiments. (B,C)
Requirement of Spry Y201. (B) Section through eye of w;
sev-GAL4/UAS-spry fly that expresses Spry under control of
sev-GAL4 in developing eye. Ommatidia are disorganized and
some (arrowheads) are missing photoreceptors. (C) Similar section of w;
sev-GAL4/UAS-spryY201F that expresses
SpryY201F under control of sev-GAL4. Ommatidia
appear normal. (D) Binding of Spry to substrate-trapping Csw in S2
cells. Whole cell lysates were prepared from transfected S2 cells expressing
Csw (lanes 1, 3) or CswC583S, a substrate-trapping form of the
enzyme (lanes 2, 4). Aliquots of lysates were directly resolved by SDS-PAGE
(lanes 1, 2) or first immunoprecipitated with anti-Csw (lanes 3, 4).
Immunoblots were probed with anti-Spry or anti-Csw as indicated. More Spry
co-immunoprecipated with CswC583S (lane 4) than with Csw (lane 3).
Similar results were obtained in three experiments. (E) Binding of Spry
to substrate-trapping Csw in imaginal discs. Eye-antennal discs dissected from
third instar transgenic larvae expressing myr-Csw, myr-CswC583S or
myr-CswG547E, a dominant-negative Csw that does not function as a
substrate trap, were homogenized and directly resolved by SDS-PAGE (lanes 1-3)
or immunoprecipitated with anti-Csw and then resolved by SDS-PAGE (lanes 4-6).
Immunoblots were probed with anti-Spry or anti-Csw as indicated. Positions of
Spry42 and Csw are shown; Spry70 is variably detected in eye disc lysates.
More Spry42 co-immunoprecipitated with myristylated CswC583S than
with myristylated Csw or CswG547E. A similar result obtained in a
repeat experiment, except in this case Spry70 isoform predominated in
co-immunoprecipitate.
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