lecture 7 Flashcards
cerveisiae life cycle
- after spindle pole body duplication, bud emergence
- bug dets progressively larger and then chromosome segregation and cytokinesis
- produces a mother celll (regular size) and a much smaller daughter cell that will get larger and grow and then undergo the same process
- spends longer in g1
sombe life cycle
fission yeast
- starts with DNA replciation
- rod gets longer and longer
- formation of septum (cytogenesis occurs through this)
- divides into two equal sized cells
- distinct moropholical features at every stage can be used to idnietify what stage of cell cycle its at.
- spends longer in g2 and M phases
cdc
cell division cycle mutants, both cerevisae and pombe have temperature sensitive mutants which cause defects in spefici proteins required to progress through the cell cycle.
loss of cdc 2 activity in s. pombe
prevents entering into M phase
cdc 2
a cyclin dependent kinase (CDK)
cdc 2 in cerevisiae = cdc ____ in pombe
28
are proteins controlling cell cycle highly conserved between eukaryotic organisms
yes; human CDKs can be used in yeast cells with mutations to gain functional CDKs
kinases
- put phosphate gorps on
phosphatases
remove phosphate groups from proteins
MPF
- heterodimer of CDK an mitotic cyclin (cdc 2)
cdc 2 activity
- is not active, phosphorylation activity remains low at G1-S phase and increases towards G2 phase, peaking at the start of mitosis
cdc 2 presence/amount
- remains constant
what does a western blot of the cdc 2 and cyclin samples show?
- mitotic cyclin bands are greater at one point in cell cycle and disappear and then reappear. (peals at G2,drops off during mitosis and then increases and drops off at that point gain)
- cdc 2 bands remain constant
does cyclin have enzymatic activity
- no but it associates with a kinase that does
activity of mitosis promoting factor
- peaks at late g2, and drops off
- this correlates with levels of cclin
what is the dotted line
it is the level of cyclin (red is the activity of cdc 2)
- beings low in g1 and then increases and peaks in late G2 and then drops off
- having more cyclin present may turn on cdc atvitiy
when does MPF form
- late g2, cdc 2 binds cyclin to form and activate MPF
why is it that there is a lot of cyclin in s phase but no cdc 2 activity
more than one type of cyclin? there must another level of regulation?
when are cyclins presnt
- ## at the cell cycle stage that they trigger and are absent in other cell cycle stages.
what are the types of cyclin
G1, G1/S, S and mitotic
how are cyclins regulate
- transcription factors that cn turn on at diff phases of the cel cycle
- ubiquitin mediated proteasome dependent degradation
what is ub-mediated proteasome-dependent degradation
- degrades the cyclins when no longer needed
ub
76 amino acid protein
- found in all eukaryotic clls
polyubiquitination
- of a target protein acts as a signal for the targeted protein to be degraded by the proteosome
- proteins are now directed towards “garbage can”
- degradative enzymes isnide
- ate hydrolysis required to move the protein and ub to proteasome for degradation
- all cyclins are destroyed at certain phases of the cell cycle
what happens t ub proteins when autophagy doesn’t work
- acclamation of UB proteins
SCF uniqutuinin ligaes
- target cyclins for destruction by ub at certain phases of the cell cycle
is ub degraded in the proteosome
no
wee1
kinase phpsorylates CDK, causing it to be activated adds phosphate group at tyrosine pos 15
cdc25
phosphatase that removes the inhibitory phosphate group from CDK and promote sits activation
what regulates MPF activty
wee1 and cdc25 by addition ore removal of phosphate groups on CDK, removes the phosphate pout on by wee1 kinase, allowing MPF (cyclin idk complex to be fully activated)
mutation in wee1 kinase or excess of cdc 25
- phosphate group not added to CDK
- increase in mitotyic cyclin during s pace and g2
- premature trigger tino mitosis (cells dot expand and elongate), small cells because of decreased G2 no break from addition of phosphate group
- cell enters mitosis too early
same phenotype as if there was an excess of cdc 25 (removing the phsppahte group right away)
mutation in phpshatase so that it is no longer functional
- phosphate group remains on
- cell grows and grows but does not. have signal to enter mitosis
- elongated cells that are stuck in g2 and cannot go to mitosis
this might also happen if too much/too active wee1 kinase putting on phosphate groups
another Layer of regulation after cdc 25 removes phosphate grou
- CAK puts a phosphate group at threonine 161 on CDK
what are all the components needed for MPF to be active
- sufficient mitotic cyclin synehtsized
- associate it with CDK
- inactivation phspph group at tyr 1,5 removal by cdc 25, and then
- acting phosphate grou by CAK at thr 161
CAK
an activating kinase
active MPF rols
- chrom condenation, disassembly of nuclear envelope, interpose microtubule dissamebly and mitotic spindle formation (MAP)
- remodeling Golgi, er
basically CDK kinase phosphorylation leads to the proomtoion of cell to enter mitosis
when does inactivation of MPF
- inactivation at anaphase needed
mechanism for inactivating MPF
- rapid mitotic cyclin degradation after polyuiquintainon
- requires APC (anaphase promotic complex/cyclosome)
APC
ub ligase active at end of mitosis.
- sticks on ub molecules onto mitotic cyclin to promote degradation of the cell
how do ub ligases (like APC) know where to stick on ub
- ## destruction box (nine amino acids found in cyclins), helps promote rdestrucftion of proteins by the proteosome, and recognition by ligases
is CDK polyubiqtuianteed ?
NO but cannot have high activity without the mitotic cyclin after anaphase
