Program of the 2001 International Worm Meeting - Sternberg Lab ...

925
925. CaM-K signal cascade of C.
elegans functionsin vivo
Yoshishige Kimura 1 , Hiroshi
Tokumitsu 2 , Masatoshi Hagiwawa 1
1 Medical Research Institute, Tokyo Medical
and Dental University
2 Kagawa Medical University
Ca 2+ /CaM protein kinases (CaM-K) have
significant roles for Ca 2+ -dependent signal
transduction. CaM-K-mediated signal cascade
consists of the upstream CaM-kinase kinase
(CaM-KK) and downstream CaM kinase I
(CaM-KI) and CaM-kinase IV (CaM-KIV). A
transcription factor cAMP-responsive element
binding protein (CREB) is known to be
activated by phosphorylation with downstream
CaM-KIV.
In this study, we identified C. elegans
orthologues of mammalian CaM-KK (ckk-1),
CaM-KI (cmk-1) and CREB, and found that
CaM-KK-CaM-KI-CREB cascade is conserved
and operated both in vitro and in transfected
cells. Furthermore, we investigated the
physiological functions through this signal
cascade by genetical approaches. The
distribution of activated CREB was analyzed by
using the transgenic worms that carries
4XCRE::gfp fusion gene. GFP fluorescence
cannot be seen in normal development,
however, transgenic worms carrying
constitutive-active form of cmk-1 induces
GFP-expression in small number of sensory
neurons in head and tail. On the other hand,
wild type and mutant cmk-1 at T179A did not
show any CREB activation, suggesting the
requirement of Ca 2+ mobilization and the
upstream kinase which was identified as
CaM-KK . In addition, CREB-deficeient worms
show no GFP fluorescence reasonably.
Interestingly, when the worms were maintained
in starved condition, the number of
GFP-positive neurons increased and the
intensity of GFP fluorescence was dramatically
enhanced. These results suggest that
CaM-KK-CaM-KI-CREB cascade of C. elegans
is conserved in vivo in the some sensory neurons
and activated by the circumstantial stimuli.
926. Guanylate Cyclase Beta 2
Homologues in C. elegans
David Karow 1 , Jennifer Chang 2 ,
Scott Nicholls 2 , Ronald E. Ellis 3 ,
Martin Hudson 4 , David Morton 4 ,
Michael Marletta 5
1 Cellular and Molecular Biology, University of
Michigan, Ann Arbor, MI 48109
2 Howard Hughes Medical Institute, University
of Michigan, Ann Arbor, MI 48109
3 Department of Biology, University of
Michigan, Ann Arbor, MI 48109
4 Department of Biological Structure and
Function, Oregon Health Sciences University,
Portland, OR 97201
5 Cellular and Molecular Biology, Howard
Hughes Medical Institute, University of
Michigan, Ann Arbor, MI 48109
926
Soluble guanylate cyclases (sGCs) catalyze the
conversion of GTP to cGMP. In mammals, the
most well-studied sGC is a heme-containing,
heterodimer, composed of α1 and β1 subunits.
Nitric oxide (NO) binds to the heme, which is
ligated to the β1 subunit, and stimulates the
activity of the enzyme 400-fold. A β1
homologue, β2, has been cloned from rat and
human kidney cDNA libraries. This subunit
appears to be expressed in the kidney collecting
duct cells, but its heme binding status,
sensitivity to NO, and intracellular localization
are unknown. We want to elucidate the
structure and function of this protein.
Analysis of the C. elegans genome
predicted seven putative sGC β subunits,
including five β2 homologues, (gcy-32, gcy-34,
gcy-35, gcy-36, and gcy-37). We cloned these
five homologues, by RT-PCR and RACE, and
found that each of them contains the residues
necessary for GTP binding, catalysis and heme
binding. These findings suggest that the β2
homologues in C. elegans are receptors in a
cGMP-dependent signaling system.
Our data are consistent with this
hypothesis. Preliminary characterization of the
bacterially-expressed N-terminus from gcy-35
suggests that it can bind heme. Promoter::green
fluorescent protein (GFP) fusion studies localize
all of the β2 homologues to four candidate
sensory neurons (URXL, URXR, AQR and