Abstract:
Screening a cDNA expression library with a radiolabelled cal-
modulin (CaM) probe led to the isolation of AtCaMRLK, a
receptor-like kinase (RLK) of
Arabidopsis thaliana
. AtCaMRLK
polypeptide sequence shows a modular organization consisting
of the four distinctive domains characteristic of receptor kinases:
an amino terminal signal sequence, a domain containing seven
leucine-rich repeats, a single putative membrane-spanning seg-
ment and a protein kinase domain. Using truncated versions of
the protein and a synthetic peptide, we demonstrated that a region
of 23 amino acids, located near the kinase domain of AtCaMRLK,
binds CaM in a calcium-dependent manner. Real-time binding
experiments showed that AtCaMRLK interacted
in vitro
with
AtCaM1, a canonical CaM, but not with AtCaM8, a divergent iso-
form of the Ca
2
+
sensor. The bacterially expressed kinase domain
of the protein was able to autophosphorylate and to phosphorylate
the myelin basic protein, using Mn
2
+
preferentially to Mg
2
+
as an
ion activator. Site-directed mutagenesis of the conserved lysine
residue (Lys
423
) to alanine, in the kinase subdomain II, resulted in
a complete loss of kinase activity. CaM had no influence on the
autophosphorylation activity of AtCaMRLK. AtCaMRLK was
expressed in reproductive and vegetative tissues of
A. thaliana
,
except in leaves. Disruption in the AtCaMRLK coding sequence
by insertion of a
DsG
transposable element in an
Arabidopsis
mutant did not generate a discernible phenotype. The CaM-
binding motif of AtCaMRLK was found to be conserved in several
other members of the plant RLK family, suggesting a role for
Ca
2
+
/CaM in the regulation of RLK-mediated pathways.
