Protein Kinases

what can be identified or suggested by amino acid sequence motifs?

protein functional characteristics

Amino acid domains

structural units with specific functions, typically encoded by a single exon

Phosphatases

Enzymes that reverse kinase action

Protein kinases

Enzymes that phosphorylate proteins, typically involved in signaling cascades.

How many kinases and phosphatases does the human genome encode?

around 520 kinases and 150 phosphatases

Serine/threonine kinases

phosphorylate serine or threonine residues

Bioinformatics and kinases

identified over 500 kinase-encoding genes and key catalytic residues using sequence alignment

PKA structure

heterodimer of 2 regulatory (R) subunits and 2 catalytic (C) subunits

What happens to PKA as [cAMP] increases?

cAMP binds R subunit → conformational change. Complex dissociates releasing C subunits

How is the cAMP binding site of PKA characterised?

GxGxxG sequence motif

Glucagon

activates PKA in response to low blood glucose levels

What happens when PKA phosphorylates glycogen synthase?

GS converted to its inactive b form

Glycogen phosphorylase

activated by PKA → glycogen breakdown and glucose release

How many glycogen molecules are broken down as the result of 1 glucagon?

10^8, showing kinase signalling has massive amplification effects

How is PKA activated

• ligand binding to GPCR

• adenylyl cyclase activated (ATP → cAMP)

• cAMP activates PKA

• response

Kinase active site residue arrangement

characteristic and conserved eg DFG motif at active site

general kinase mechanism

• ATP binds kinase active site

• substrate binds active site

• gamma phosphate ATP → Ser/Thr/Tyr

• substrate released

• ADP released

Activation of kinases

Often involves unmasking the active site through activation loop displacement or via pseudosubstrate domains

Activation loop

structure that blocks the active site until its phosphorylation causes conformational change

Protein Kinase C (PKC)

A superfamily of homologous kinases made up of domains

Pseudosubstrate domain

region that keeps PKC inactive by binding to the substrate-binding cavity until DAG binds the C1 domain

where is the pseudosubstrate domain found?

very close to domain being phosphorylated

pseudosubstrate domain structure

short AA sequence lacking serine/phospho-acceptor residues

Target specificity

Kinase targets have similar sequences around phospho-acceptor sites eg arg xx ser motif

What does phosphorylation by kinases change in a protein?

localisation, interactions by affecting affinity, half-life and sensitivity to signals

AMP activated protein kinase activation

activated by allosteric mechanism stimulated by reduced ATP:AMP/ADP ratio

AMPK alpha subunit

contains kinase domain and residue Thr172 which is phosphorylated by upstream kinase (LKB1 complexed with STRAD and MO25)

AMPK beta subunit

carbohydrate-binding module allowing glycogen association

AMPK gamma subunit

enables response to changes in AMP:ATP as it contains 4 domains which bind adenine nucleotides

glucose pathways activated by AMPK

phosphorylates targets in glucose transporter trafficking to increase GLUT4 localisation, phosphorylates PFKB3 to increase activity of glycolysis enzyme PFK1