Cell Cycle Control 
James L. Mailer, Ph.D. — Investigator 
Dr. Mailer is also Professor of Pharmacology at the University of Colorado School of Medicine. He received 
his B.S. degree in biochemistry from Cornell University and his Ph.D. degree in molecular biology from 
the University of California, Berkeley, where he worked with John Gerhart. He then carried out postdoc- 
toral studies with Edwin Krebs at both the University of California, Davis, and the University of Wash- 
ington before moving to Colorado. 
TWO events mark the reproductive life of a 
cell: replication of the DNA and its distribu- 
tion to daughter cells at mitosis. Because of the 
central importance of cell reproduction to or- 
dered cell growth, cells have evolved rigorous 
controls to ensure that both processes are carried 
out with high fidelity and at the appropriate time. 
My laboratory is interested in understanding the 
nature and regulation of these controls with re- 
spect to how a cell commits itself to replicate its 
DNA and how it knows when to divide. The deci- 
sion to get ready for DNA synthesis is made in the 
Gi period (after mitosis but before DNA synthesis 
or S phase), and the decision to begin cell divi- 
sion (M phase) is made in G2 (after S phase is 
complete) . There is abundant evidence that these 
decisions are made at checkpoints or restriction 
points in the cycle. The nature of these Gi and G2 
decision-making periods in the cell cycle under- 
lies fundamental processes operative in early em- 
bryonic development and in malignancy. 
G2 M Regulation 
In the last three years great strides have been 
made in understanding the checkpoint governing 
entry into mitosis. For many years it had been 
known that mitosis is dominant over interphase. 
That is, the signal that tells a cell to go into mito- 
sis, if inappropriately expressed earlier in the cy- 
cle, can cause premature mitosis and cell death. 
Our laboratory developed an interphase cell ex- 
tract from frog eggs in which addition of mitotic 
signals caused synthetic nuclei in the extract to 
enter mitosis in vitro. We then purified the mito- 
sis-signaling enzyme (called M-phase factor or 
MPF) and found that it was composed of a protein 
kinase complexed to a G2 cyclin. Kinases have 
the ability to attach a phosphate group to many 
different cellular proteins, modifying their func- 
tion and causing profound changes in cellular 
biochemistry. The protein kinase subunit of the 
enzyme that catalyzes mitosis was identified as a 
vertebrate homologue of the cdc2 gene, which 
had been genetically implicated in the control of 
mitosis by the study of certain mutants in yeast. 
G2 cyclins are proteins that accumulate during 
interphase, reach high levels in late G2 phase, and 
are then degraded near the metaphase/anaphase 
transition in mitosis. This degradation is required 
for cells to complete mitosis successfully and 
enter Gi. In most cells there are two classes of G2 
cyclins, termed A and B cyclins, that differ in se- 
quence similarity and have different kinetics of 
accumulation and degradation. Both bind cdc2 
kinase, but A-type complexes are activated much 
earlier in the cell cycle than B-type complexes. 
To study the differences in A- and B-type cyclins, 
we have expressed the frog cyclin genes in insect 
cells. This allows us to make large quantities of 
functionally active frog cyclins, which are able to 
drive synthetic nuclei into mitosis in extracts 
after complexing with cdc2 kinase. 
We are interested in the mechanism of activa- 
tion of MPF in oocytes during the cell cycles of 
meiosis I and II. In these cycles the synthesis of 
proteins other than cyclin is required for activa- 
tion of MPF. One protein required for meiosis I 
and II is the product of the mos proto-oncogene. 
Proto-oncogenes are the normal cellular counter- 
part of mutated oncogenes found in cancer cells, 
suggesting that they act by perturbing normal cel- 
lular pathways. In general, very little is known 
about how proto-oncogenes work, but the spe- 
cific involvement of mos in cell cycle control is 
the clearest example of a specific function for any 
proto-oncogene in a defined cellular process. 
The mos gene encodes a serine/threonine pro- 
tein kinase, indicating that a substrate for phos- 
phorylation by mos exists that can lead to activa- 
tion of MPF as well as stabilization of cyclin in 
meiosis II. This year we discovered that B-type 
Xenopus cyclins were mos phosphorylation sub- 
strates and that the affected sites were distinct 
from those phosphorylated by cdc2 kinase itself. 
As soon as the exact site of mos phosphorylation 
in cyclin is determined, we can ablate the site(s) 
and evaluate effects on cyclin function and cell 
cycle control. 
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