Protein kinases and phosphatases Flashcards
What is the most common PTM?
phosphorylation by a kinase
Why is the rxn carried out by protein phosphatase not the opposite of phosphorylation by a kinase?
because ATP is not masde when phosphaet is removed from the protein
Which ion facilitates the breaking of the beta and gamma phosphate bond during protein phosphorylation?
magnesium
How are kinases different from phosphorylases and pyrophosphorylases?
The substrates of these enzymes are small molecules, not proteins as they are for kinases. Kinases also only use ATP (and occasionally GTP), while the phosphorylases utilize other nucleotides.
List the functions that are elicited by protein kinases.
- transcriptional regulation
- cell structure and motility
- desensitization of receptors by binding beta-arrestin
- facilitation of exocytosis
What are the different types of kinases?
two protein kinase families:
- serine-threonine
- tyrosine
What are the structural features of protein kinases?
regulatory and catalytic components, normally within a single polypeptide. Domains can be on either end of the polypeptide. In some cases, like RTKs, the regulatory domain is a ligand-binding domain.
Where on the kinase protein does the part of the protein being phosphorylated sit? The ATP molecule?
Protein residue being phosphorylated sits in the substrate-binding loop. ATP sits in the phosphate-binding loop.
What are the parts of the protein kinase catalytic domain?
3 parts:
- ATP-binding loop
- catalytic loop (transfer between phosphate and hydroxyl group of ser/thr/tyr
- substrate-binding loop
Describe the residues of the protein kinase active site
- negatively charged aspartate allows for binding to the peptide substrate
- positively charged lysine allows for binding to the phosphate molecule
How does a kinase know to add a phosphate to either a ser/thr or to a tyr?
- phosphorylation site consensus sequence around residue being phosphorylated (ser/thr residues are surrounded by arginine, tyr residue is surrounded by arginine on one side and glutamate on the other)
- cleft size of the kinase active site (ser/thr are smaller residues and so fit into more shallow clefts. Tyr is a larger residue and fits into a deeper cleft)
Examples of ser/thr kinases and tyr kinases.
ser/thr:
- protein kinase A, G, C
- CaM kinase
tyr:
- EGF receptor
- Src
- RTK
Regulation of protein kianse A?
PKA is a heterodimer of two polypeptides when inactive: regulatory subunit and catalytic subunit. Cyclic AMP binds the regulatory subunit, releasing active catalytic subunit.
It is inactive when bound to regulatory subunit because this subunit acts as a pseudosubstrate (similar peptide sequence to substrate) so is autoinhibitory.
Describe PKC activation and regulation.
- regulatory and catalytic domains are within the same polypeptide
- in catalytically incompetent, inactive state, the protein is folded over so the catalytic domain is inhibited by autoinhibitory domain of regulatory domain
- phoshorylation of catalytic subunit by PDK1 activates autophosphorylation activity of catalytic domain
- autophosphorylation of the catalytic domain makes it catalytically competent, but it is still folded over and inactive
- calcium then binds to regulatory domain, allowing for docking to the plasma membrane via phospholipid.
- membrane docking allows for access to DAG. Binding to DAG facilitates opening of the catalytic domain so the substrate can dock. This final state is unfolded and active.
Describe PKB/Akt regultation.
- activated by RTK
- PKB has plextrin homology domain (PHD) that allows it to dock to phospholipid in the plasma membrane
- membrane docking puts PKB in close proximity to PKD1 which phosphorylates catalytic and regulatory domains of PKB.
- after phosphorylation, PKB dissociates from membrane in its active form.
Describe CaM kinase activation and regulation.
It is similar to PKC, but requires both calcium and calmodulin to remove the autoinhibitory domain from the catalytic domain, partially activating it. When calmodulin is bound in all four sites by calcium, it can fold over and bind to proteins with calmodulin binding sites
- cam kinase is fully activated when catalytic domain is autophosphorylates the regulatory domain so it cannot re-inhibit cat domain
- calmodulin is no longer needed, so it leaves reg domain. cam kinase remains active as long as the reg domain stays phosphorylated
describe RTK regulation.
requires dimerization for autophosphorylation. often bound by dimerized ligand. each phosphorylation site within the phosphorylation domain is a unique binding site for a cytosolic protein (signal divergence!). These transducers are necessary because RTK is stuck in the membrane.
List some non-receptor tyrosine kinases.
- SRC
- JAK
What is Src? Describe its activation and regulation.
A non-receptor tyrosine kinase.
- intracellular and membrane-bound by myrositoylation
- catalytic domain is locked into reg domain, inhibiting it
- binding of regulatory proteins to SH2 and SH3 reg regions allows for PTK catalytic domain to have one residue phosphorylated and the other dephosphorylated, thus activating it
Describe the steps of JAKs STATs signaling.
- ligand binds to non-tyrosine kinase receptor on the outside of the cell
- receptor dimerizes, activating JAK
- JAK is autophosphorylated, and then phosphorylated the intracellular portion of the receptor so it has phosphorylation residues to which proteins can bind and become activated
- STATs are recruited to these phosphate groups, and docks to them
- when docked, JAKs can phosphorylate STATs, causing them to release from phosphate docking site and dimerize to each other
- STATs dimer than translocates to the nucleus where it regulates txn
How is the JAK STAT pathway regulated?
- members of the SOCS family bind to JAK and prevent it from binding the receptor
- members of the PIAS family bind STAT in the nucleus and prevent its association with DNA
How is MAPK activated?
duel-activated kinase
- MKK is phosphorylated by Raf, activating it and releasing from ERK
- ERK then dimerizes and is phosphorylated at 4 sites, tyrosines and threonines
- activated ERK dimer heads to the nucleus where it acts as a ser/thr kinae
describe protein phosphatase 2A (PP2A).
- a stable heterotrimer
- regulatory subunit brings different substrates in close proximity to catalytic subunit
- scaffolding subunit holds everything in place
- interaction with microtubules
