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REVERSIBLE PROTEIN PHOSPHORYLATION 271
CDKs and cyclins in kinetoplastids
A number of genes for CDK-like kinases have
been identified in protozoan parasites. However,
the exact physiological roles for many have
yet to be determined. Since parasitic protozoa
have complex life cycles, different CDKs might
be used to regulate stage-specific events. For
example, trypanosomatids contain a family
of CDK-related kinases referred to as CRKs.
L. mexicana CRK1 (LmmCRK1) has significant
homology with mammalian and yeast
CDK2s (CDC28 in budding yeast or cdc2 in
fission yeast). However, the PSTAIRE cyclinbinding
domain in LmmCRK1 is changed to
PCTAIRE and LmmCRK1 fails to complement
fission yeast temperature-sensitive cdc2
mutants. The activity of LmmCRK1 appears to
be post-translationally regulated since the protein
is detected at all stages, while the histone
H1 kinase activity is present only in promastigotes
and metacyclics, but not in amastigotes.
Attempts to generate the lmmcrk1 null phenotype
have been unsuccessful, suggesting that
LmmCRK1 has an essential function. The
CDK–cyclin complex also contains additional
proteins such as CKS (cyclin-dependent kinase
subunit) that interact with substrates to stimulate
kinase activity. The fission yeast homolog
of CKS is known as p13 suc1 . LmmCRK1 does
not interact with the fission yeast p13 suc1 .
However, another CDK-related leishmanial
kinase, LmmCRK3, which contains PQTALRE
instead of the PSTAIRE sequence binds
to fission yeast p13 suc1 and the Leishmania
homolog p12 cks1 . The association of CRK3 with
p13 suc1 or p12 cks1 is reduced in extracts from
metacyclic forms (cell cycle-arrested) compared
with promastigote or amastigote forms.
This reduced binding also correlates well with
histone H1 kinase activity. LmmCRK3 also
appears to be essential since attempts to disrupt
its locus result in an increase in ploidy. The
LmmCRK3 kinase activity of promastigotes
appears to be required mainly during the G 2 /M
transition.
Three well studied T. brucei CRKs have substitutions
in the canonical PSTAIRE sequence.
TbCRKs 1–3 contain PCTAIRE, PSTAVRE, and
PQTALRE motifs, respectively. An additional five
CRK genes have been tentatively identified in
T. brucei. TbCRK4, unlike other CRKs, contains
large insertions in the catalytic domain and is
expressed at all life-cycle stages. The kinase
domain of TbCRK5 has strong similarity with
MOK (MAPK/MAK/MRK overlapping kinase).
Three cyclin genes have been identified in
T. brucei. CYC1 was initially thought to be a
mitotic cyclin. However, recent reports indicate
that the original sequence was incorrect and
CYC1 does not exhibit mitotic cyclin characteristics.
Two additional T. brucei cyclin genes,
CYC2 and CYC3, have been isolated by complementation
of yeast G1 cyclin mutants. T. brucei
appears to contain eight cyclin homologs
(CYC2–9). CYC2 is related to the budding yeast
PHO80 cyclin. The ty-epitope tagged CYC2
associates with L. mexicana p12 cks1 . Histone H1
kinase activity was detected in tyCYC immunoprecipitates,
suggesting its role in the formation
of an active CDK–cyclin complex. TbCYC3
shows homology to mitotic B-type cyclins.
Both TbCYC2 and TbCYC3 are expressed in
all stages, and an interaction of TbCRK3 with
both TbCYC2 and TbCYC6 has been demonstrated
by yeast two-hybrid screening. TbCYC6
showed significant sequence similarity with
mitotic cyclins, and complements budding
yeast CLN123 mutant. Functional knockout
of TbCYC6 with RNA interference (RNAi)
demonstrated that TbCYC6 is essential for
mitosis in both procyclic and bloodstream
forms of the parasite. Interestingly, in procyclic
forms, the absence of TbCYC6 causes cytokinesis
in the absence of mitosis. Consequently,
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA