trans

254 INTRACELLULAR SIGNALING
the knockout of both CUB alleles only occurred
in cells that were simultaneously transformed
with a functional copy of the gene. The inability
to knock out both alleles in the absence of gene
replacement demonstrates that CUB is essential
to parasite survival. Its function is unknown.
Kinetoplastids also contain an EF-hand
protein that is associated with the flagellum,
that was initially described in T. cruzi as FCaBP,
and in T. brucei as Tb17. FCaBP is tandemly
repeated within the T. cruzi genome and
exhibits slight variations between different
strains. By contrast, T. brucei contains four distinct
members of the FCaBP group (including
Tb17) that have been collectively referred to as
calflagins. FCaBP has four EF-hands of which
two at the carboxyl terminus appear to bind
Ca 2 with high affinity. FCaBP is of unknown
function but is acylated at the amino terminus
by myristic acid and palmitic acid. These posttranslational
modifications appear to be important
for its flagellar location. A Ca 2 -acyl switch
may allow FCaBP to reversibly associate with
membranes and regulate enzyme activities.
To date, binding partners for FCaBP/calflagins
are not known. However, FCaBP resembles
the acyl-switch protein recoverin of sensory
flagella in mammalian cells. In the presence of
Ca 2 , recoverin binds to and inhibits rhodopsin
kinase. It is not yet known why the flagellum
of kinetoplastids contains different EF-hand
family members.
Apicomplexans provide further evidence of
the diverse structures that incorporate the
EF-hand motif. A family of calmodulin-like
domain protein kinases (CDPKs) is found in
P. falciparum and T. gondii. While typical Ca 2 -
sensitive kinases associate reversibly with
calmodulin, the CDPKs possess their own EFhand
motifs. Similar kinases are found in plants.
In P. falciparum, CDPK1 associates with sites of
membrane fusion, and may be associated with
the invasion process. By contrast, PfCDPK3 is
expressed specifically in the sexual erythrocytic
stage where it may mediate Ca 2 -sensitive steps
of gametogenesis. In T. gondii, a similar situation
exists, except that only two members
of the CDPK family have been identified in
the genome (TgCDPK1 and TgCDPK2). Only
TgCDPK2 is expressed in tachyzoites. An
inhibitor of Ca 2 -sensitive protein kinases
KT5926 blocks the activity of TgCDPK2, prevents
phosphorylation of specific parasite proteins,
and also blocks motility and attachment
to host cells.
In E. histolytica, a novel protein with EFhands
has been identified along with calmodulin;
the protein is called EhCaBP. It is not found
in E. invadens. Binding partners for EhCaPB
have been detected by affinity procedures,
and the subset of proteins that associate with
EhCaBP in trophozoites is different from the
binding partners for calmodulin. In keeping
with that observation, EhCaBP is able to activate
a novel plant protein kinase, while calmodulin
cannot. Additionally, two EF-hand proteins
have been found associated with cytoplasmic
granules. Each of the proteins, called granins 1
and 2, is predicted to contain three functional
EF-hands. Since granule discharge may be triggered
by Ca 2 , the granins, along with calmodulin
and EhCaBP, may help regulate exocytosis
in these organisms. Ca 2 -sensitive discharge
of dense granules from Entamoeba has been
reported.
Taken as a whole, it is evident that along with
calmodulin, parasites contain a wide range of
EF-hand proteins. Many of these proteins likely
interact with targets that are not present in the
mammalian host. Plasmodium and Toxoplasma
contain a variation on that theme by utilizing
proteins whose catalytic domain is fused to
the EF-hand Ca 2 sensing domain. In addition,
other calcium-binding proteins exist in these
cells. Inducible RNAi systems, DNA microarrays
and proteome analyses will go a long way
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA