Chapter 6 Protein Phosphorylation

Objectives Know the general enzymes involved in phosphorylation Know the general enzymes involved in dephosphorylation Know the other types of covalent modifications Understand the general mechanisms of phosphorylation/dephosphorylation pathways

Phosphorylation Control

Phosphorylated Amino Acids Only occurs on S/T/Y – S/T kinases – Y kinases Can happen to multiple amino acids in the same protein Can have multiple kinases doing the work on the same protein

Properties of Kinases Rapid reactions (less than a second) Amplification Controlled by second messengers Casein kinases not controlled by second messengers Specificity – Some very specific – Other will phosphorylate many proteins

Serine / Threonine Kinases Many examples Diverse functions Possible oncogenes

cAMP-dependent protein kinase cAPK or PKA (protein kinase A) – Controls: Metabolic rates – Glycogen breakdown Gene expression – CRE-binding protein (CRE response element) – Activated by cAMP – Tetramer Two regulatory subunits Two kinase (catalytic) subunits

cAMP-dependent protein kinase cAPK or PKA (protein kinase A) – Invariant amino acids in catalytic domain K 47, 72, 76 G 50, 52, 55 – Consensus phosphorylation site RRxSx RRxTx – K m = μM – V max = 8-20 μmol/min/mg

PKA

PKG Protein Kinase G – Activated by cGMP – Almost the same as PKA but with binding to cGMP – Regulatory and catalytic subunits – Can also bind cAMP (higher concentration needed)

PKC At least 10 isoforms Monomeric – Regulatory and catalytic domains Contain up to 4 Zn for structural reasons Controlled by – Ca, phospholipids (DAG, InsP 3 ) Calpains role? Activation or degradation Consensus sequence – S/T-X-R/K

Ca 2+ -calmodulin-dependent protein kinases CaM kinase – Activated by calcium – Alpha, beta, and beta’ subunits All have kinase activity Activated by autophospho rylation – Wide variety of substrates

G-protein coupled receptor kinases GPCRK –β ARK (beta-adrenergic receptor kinase) – Binds to βγ subunit of heterotrimeric g protein (PH domain) Targets back to GPCR to phosphorylate receptor – Only ligand bound receptors are phosphorylated – Turns off signaling

Protein Kinase B PKB or c-Akt – V-Akt is oncogene – Similar to PKA or PKC – Less known, but has a PH domain – Metabolic regulation

Heme regulated protein kinase Important for hemoglobin synthesis regulation – If heme is absent Kinase phosphorylates eIF-2 stopping GEF activity – If heme is present Kinase not active, protein synthesis occurs Other protein synthesis pathways controlled this way – Interferon

Tyrosine Kinases Less common to have pY than pS or pT Consensus sequence – R/K-X-X-D/E-X-X-X-Y

Receptor Tyrosine Kinases Extracellular domain for ligand binding Single transmembrane domain Cytosolic domain with kinase activity – Can phosphorylate multiple substrates Phosphatidylinositol 3 kinase, GTPase activating protein, G-proteins (not heterotrimeric G proteins) – Can autophosphorylate Self, or another RTK – Usually dimerize

GRB2 SOS –P P– RAS RTK pathway

Non-Receptor Tyrosine kinases Cytosolic proteins with tyrosine kinase activity – Janus kinase – STAT

Mitogen Activated Protein Kinases ERKs (extracellular signal-regulated kinases) MAPs (mitogen-activated protein kinases) They are S/T kinases – Can translocate to the nucleus and phosphorylate transcription factors – Activated by T/Y kinase MAPKK (MAP/ERK kinase or MEK) MAPKK is activated by MAPKKK or MEKK

MAPK pathway Signals can originate from RTKs, G-proteins, heterotrimeric g- proteins Different pathways rely on scaffolding proteins

GRB2 SOS Raf –P P– RAS MEK –P MAPK –P To the nucleus!! MAPK pathway

Histidine Phosphorylation Histidine and aspartic acid can be phosphorylated in lower organisms – Bacteria, slime mold, yeast – Little evidence of these kinases in mammals

Phosphatases PPx – Protein phosphatase 1, 2a, 2b, 3, 4, 5 are S/T phosphatases – Takes off phosphates, but also is regulated Inhibitor proteins (controlled by phosphorylation) Ca 2+ Mg 2+ PTP – Protein tyrosine phosphatase – Cytosolic – Receptor type

Other Covalent Modifications Protein cleavage – Not reversible Adenylation – Controls nitrogen metabolism Fatty acid / isoprene addition – Membrane targeting Reactive oxygen or nitrogen species (ROS, RNS) – Effect protein function

Ubiquitination