2.  After graduating from the Johns Hopkins University Biotechnology
Program, you obtain a nice, cushy job with an evil, price gouging
pharmaceutical company in their drug development laboratory.  In their
search for new, and improved anti-neoplastic chemotherapeutic drugs,
they have stumbled across a drug that they believe may interfere with
the binding of the Cyclin D and E proteins to the Cyclin Dependent
Kinases.   From your lectures in Advanced Cell Biology II, you learned
about cell cycle regulatory proteins, so you are already thinking
about that new BMW you will buy with those bonus dollars and IPO
profits.  Your assignment from the laboratory chief is:

 
B.  What observations with respect to other cell cycle related
proteins and cell morphology would convince you that they are correct
in their assumptions?

C.  How would you determine the precise region or domain of the
cyclins this drug was binding to, if you had a   14C labeled drug?

Hi -

B) Interference of cyclin D and E binding to CDK's would be expected
to prevent the cyclins from exerting their normal action which is to
drive forward the cell-cycle.

Proteins which you would expect to be effected by this interference
are:
-Rb is a target of CDK4 (activated by cyclin D1) which leads to it's
phosphorylation - this drug should decrease Rb phosphorylation
(detectable by immunoprecipitation, immunoblotting with an
anti-phosphate antibody) and decrease Rb-E2F binding (detectable by
coimmunoprecipitation, semi-quantitative Western blotting).

- CDK's are activated by the cyclins - the activity of these enzymes
would be expected to be decreased with this drug - in particular
cdk2/4/6 would be expected to have lower enzymatic activity.

- cyclin A production is dependent upon the activity of earlier
cyclins and as such, this drug would be expected to decrease the
quantity and activity of cyclin A

Morphological changes that one would expect:
Cells would arrest at the appropriate check-point - G1/S for cyclin D
and late G/S for cyclin E - this arrest would be easiest to detect
using FACS analysis to contrast cell size with quantity of DNA.  You
might also expect to see an increase in apoptosis as the cells are
restricted from continuing through the cell cycle.

A rather nice page from a graduate student at Penn detailing various
parts of the cell cycle signaling machinery:
http://www.geocities.com/CollegePark/Lab/1580/cycle.html

A brief PDF discussing some of the relevant proteins:
http://www.columbia.edu/cu/biology/courses/w3041/lectures/15_Cell_Cycle.pdf

A chart and some discussion from Sigma:
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Cell_Signaling/Pathway_Slides_and_Charts/G1_and_S_Phases_of_the_Cell_Cycle.html

Specific aims from a grant looking at Cyclin E in breast cancer cells
http://www.ucop.edu/srphome/bcrp/progressreport/abstracts/patho/3FB-0084.html

C) The easiest way to determine the region of the protein with a
labeled drug would be to mutagenize and/or express a series of
truncation mutants of the protein.  The mutagenized proteins could be
expressed with point mutations (or larger if need be) in residues
suspected of binding to the drug.  By immobilizing the expressed
construct and measuring the amount of radioactivity bound
versus/unbound one could obtain an idea of whether or not a residue
was important in the binding event.  Multiple residues could be
mutagenized in this fashion in order to establish the involvement of
multiple residues (ie it might be the case that mutating one residue
will not remove all of the binding, thus implicating that residue in
the process but suggesting that other residues are important as well. 
Deletion constructs function in much the same way, by truncating off
regions of the protein in a sequential fashion, from either end of the
protein (or even from the middle bits) one can obtain constructs that
are missing regions of the protein.  If after deleting a region of the
protein, your binding to drug disappears, then you've found the
critical region.

search strategy:
Google: cyclin cell-cycle
Google: "cyclin D" cell cycle
Google: "cyclin E" cell cycle