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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.

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chapter 6

Chapter 6

Protein Phosphorylation

objectives
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
phosphorylated amino acids
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
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
Serine / Threonine Kinases
  • Many examples
  • Diverse functions
  • Possible oncogenes
camp dependent protein kinase
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 kinase1
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
    • Km = 10-20 μM
    • Vmax = 8-20 μmol/min/mg
slide10
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)
slide11
PKC
  • At least 10 isoforms
  • Monomeric
    • Regulatory and catalytic domains
  • Contain up to 4 Zn for structural reasons
  • Controlled by
    • Ca, phospholipids (DAG, InsP3)
  • Calpains role? Activation or degradation
  • Consensus sequence
    • S/T-X-R/K
ca 2 calmodulin dependent protein kinases
Ca2+-calmodulin-dependent protein kinases
  • CaM kinase
    • Activated by calcium
    • Alpha, beta, and beta’ subunits
      • All have kinase activity
      • Activated by autophosphorylation
    • Wide variety of substrates
g protein coupled receptor kinases
G-protein coupled receptor kinases
  • GPCRK
    • βARK (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
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
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
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
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
rtk pathway
RTK pathway

–P

P–

–P

P–

GRB2

RAS

–P

P–

–P

P–

SOS

non receptor tyrosine kinases
Non-Receptor Tyrosine kinases
  • Cytosolic proteins with tyrosine kinase activity
    • Janus kinase
    • STAT
mitogen activated protein kinases
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
MAPK pathway
  • Signals can originate from RTKs, G-proteins, heterotrimeric g-proteins
  • Different pathways rely on scaffolding proteins
mapk pathway1
MAPK pathway

–P

P–

–P

P–

GRB2

RAS

–P

P–

–P

P–

Raf

–P

SOS

MEK

MAPK

–P

To the nucleus!!

histidine phosphorylation
Histidine Phosphorylation
  • Histidine and aspartic acid can be phosphorylated in lower organisms
    • Bacteria, slime mold, yeast
    • Little evidence of these kinases in mammals
phosphatases
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)
      • Ca2+
      • Mg2+
  • PTP – Protein tyrosine phosphatase
    • Cytosolic
    • Receptor type
other covalent modifications
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