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Chapter 6

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

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  1. Chapter 6 Protein Phosphorylation

  2. 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

  3. Phosphorylation Control

  4. 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

  5. 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

  6. Serine / Threonine Kinases • Many examples • Diverse functions • Possible oncogenes

  7. 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

  8. 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

  9. PKA

  10. 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)

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

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

  17. 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

  18. RTK pathway –P P– –P P– GRB2 RAS –P P– –P P– SOS

  19. Non-Receptor Tyrosine kinases • Cytosolic proteins with tyrosine kinase activity • Janus kinase • STAT

  20. 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

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

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

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

  24. 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

  25. 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

  26. Ubiquitination

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