Jan 30 Flashcards
What does cAMP target to start phosphorylase actviation to start glycogen degradation?
Protein kinase A
What is the phosphorylation concensus motif for phosphorylating serines?
from p-3 to P+1 position, it is pos AA-Pos AA-X-Ser-Hphobic AA
Where are most phosphorylation sites located on proteins, is conformation important?
Usually on intrinsically disordered regions, the conformation around it is not important, just needs to be on surface of protein when folded
What will a pseudokinase look like for AA sequence?
It will have all the AA’s around the posphorylation site as needed, but will not actually have, ser/thr/tyr at that site
How to determine what AA may do for kinase preference to phosphorylate protein/
Have peptides with random AA’s (except Tyr/Ser/Thr and Cys -> it can crosslink) before and after the ser/thr/tyr to be Po4’d, then make sure that one of those random AA’s is a certain something. Using radiolabelled Mg-ATP, can see if leucine is at position -5,-4…+4 where you do and don’t get phosphorylation (dark spot) as the radioactive ATP not part of PO4’d protein is washed off before scan
What do you get from global motif analysis with protein kinases?
You add drug or UV irradiation, then compared to the control you see which sites are phosphorylated more and less as a result, so get inhibited and upregulated kinases based on the motifs phosphorylated.
What is special about phsophorylase kinase (PhK)?
It has a glu instead of Ser or Thr at the site, so it is a pseudokinase
What is unique to MAP kinase?
It has a thr and tyr at the site, so it can get PO4’d twice
What happens when kinase is phosphorylated to activate it? What do pos residues of protein do and where is substrate located?
It is phosphorylated on the activation loop/T-loop. It will then shift the alpha-C helix and have a higher affinity for Mg-ATP. The substrate in the cleft between the N and C lobe will be phosphorylated using the gamma phosphate. The pos residues will lock the kinase in active conformation.
