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Protein Kinases, the Most Important Biochemical Regulatory System in Animal Cells

Protein Kinases, the Most Important Biochemical Regulatory System in Animal Cells. Big Picture Definition, History, and Gene Number Classifications Serine/Threonine Kinases Tyrosine Kinases MAP protein kinase networks and pathways. Sutherland Second Messenger Hypothesis.

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Protein Kinases, the Most Important Biochemical Regulatory System in Animal Cells

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  1. Protein Kinases, the Most Important Biochemical Regulatory System in Animal Cells • Big Picture • Definition, History, and Gene Number • Classifications • Serine/Threonine Kinases • Tyrosine Kinases • MAP protein kinase networks and pathways

  2. Sutherland Second Messenger Hypothesis

  3. Protein Kinases in Animal Cells • Cell division • Apoptosis The first messenger interacts with a receptor and a second messenger is formed

  4. History and Importance I • About 518 genes in humans encode protein kinases; there are an estimated 30,000 genes in humans, so that about 1.7% of the human genome encodes protein kinases • Protein kinases are the fourth largest gene family in humans • C2H2 zinc finger proteins (3%) • G-protein coupled receptors (2.8%) • Major histocompatibility (MHC) complex protein family (2.8%)

  5. How Many Protein Kinases Are There? (518) • The kinome refers to all protein kinases in the genome • There are 478 conventional eukaryotic protein kinases (ePKs) plus 106 pseudogenes • 388 Protein-serine/threonine kinases • 90 Protein-tyrosine kinases • 58 receptor PTKs • 32 Non-receptor PTKs • There are 40 atypical protein kinases (e.g. EF2K/alpha kinases) • 478 + 40 = 518 • The exact numbers aren’t important; understand the classification

  6. General Classes I • ACG Group • Protein kinase A; cyclic AMP-dependent protein kinase • Protein kinase C • Protein kinase G • Basic amino acid-directed enzymes that phosphorylate serine/threonine (you don’t have to memorize any sequences)

  7. General Classes II • CaMK • Calcium-calmodulin-dependent protein kinases • I • II • III • IV • Type II is a broad specificity kinase • The others are dedicated kinases with a limited substrate specificity

  8. General Classes III • CMGC • Cyclin-dependent protein kinases • These are important regulators of the cell cycle • MAP (Mitogen activated protein/microtubule associated protein) kinases • Many of these promote cell division • GSK3 (glycogen synthase kinase-3) • Clk (Cyclin-dependent like kinase)

  9. General Classes IV • PTK (Protein-tyrosine kinases) • Receptor, e.g., epidermal growth factor receptor, insulin receptor • Non-receptor, e.g., Src, Abl protein kinases • Specifically phosphorylate protein-tyrosine (note they are not tyrosine kinases but protein-tyrosine kinases)

  10. Protein Kinase Classifications • Protein-Serine/threonine • Protein-Tyrosine • Receptor: ligand binding domain and catalytic site on the same polypeptide • Non-receptor: catalytic domain separate from the receptor • Dual Specificity (both serine/threonine and tyrosine) also occur • Broad Specificity: have several substrates, e.g., PKA • Narrow Specificity: have one or a few substrates, e.g., pyruvate dehydrogenase kinase with one substrate • Classified by activator: PKA, PKG, PKC

  11. Second Messengers (Fig. 19-4)

  12. Protein Kinase and P’ase Rxns (Fig. 4-8)

  13. Reactions and Types of Protein Kinases • Be able to recognize the three amino acids with an –OH in their R-group • Serine • Threonine • Tyrosine

  14. Serine/Threonine Protein Kinases

  15. Activation of Protein Kinases • Know PKA activation mechanism • It is the only one where there is a dissociation of regulatory subunits from catalytic subunits • This was the first activation mechanism to be described, but it turns out to be atypical or unique • PKG, allosteric • PKC, allosteric

  16. Hormones and cAMP Many first messengers lead to changes in [cAMP]

  17. Protein Kinase A Structure • Bilobed • N-lobe (upper) mostly beta sheet • C-lobe (lower) mostly alpha helix • Active site between the two lobes • ATP is bound in the active site

  18. PKA Domain Structure

  19. PKA Substrate Specificity Basic-Basic-Xxx-Ser-Hydrophobic is preferred

  20. Selected Protein Kinase A Substrates • Phosphorylase kinase alpha and beta subunits • Pyruvate kinase (Liver type) • 6-Phosphofructo-2-kinase/phosphatase • Hormone sensitive lipase • Protein phosphatase inhibitor 1 • CREB (a transcription factor) • Aromatic amino acid hydroxylases • Tyrosine hydroxylase • Tryptophan hydroxylase • Phenylalanine hydroxylase • What else is special about these three enzymes? • Raf • Grandmother

  21. Earl W. Sutherland, Jr (cAMP)Edwin Krebs (PKA) Albert G. Gilman (G-protein)Edmund Fischer (PKA) Martin Rodbell G-protein)

  22. Enzyme Cascades and Phosphorylase and Synthase • Hormonal regulation • Hormones (glucagon, epinephrine) activate adenylyl cyclase • Glucagon, liver • Epinephrine in muscle • cAMP activates kinases and phosphatases that control the phosphorylation of phosphorylase and glycogen synthase • GTP-binding proteins (G proteins) mediate the communication between hormone receptor and adenylyl cyclase • Learn the regulation of the PKA-phosphorylase cascade!!!

  23. cAMP Metabolism

  24. Regulation of Glycogen Metabolism PKA  phosphorylase kinase  phosphorylase A kinase acting on a kinase that phosphorylates a protein is a cascade Steps 3 and 4 make up the first cascade to be described

  25. Protein Kinase G • Activated by cGMP • Second second messenger protein kinase (after cAMP) • Little known about physiological protein substrates despite extensive investigation • Two types of guanylyl cyclase • Second messenger generated by atrial naturetic factor as an integral membrane guanylyl cyclase • Second messenger generated by NO action on the soluble guanylyl cyclase reaction

  26. Action of cGMP Review Fig 19-23 for NO biosynthesis

  27. Cyclic GMP Metabolism (Fig. 19-24) • Integral membrane guanylyl cyclase (ANF {atrionaturetic factor} receptor) • Soluble: Activated by NO

  28. cGMP Metabolism and Action

  29. Regulation of Protein Kinases G(Equation 19.1)

  30. Selected Protein Kinase G Substrates • G substrate (cerebellum) • Vasodilator-stimulated phosphoprotein • Most substrates and their functions are unknown

  31. Calcium-Dependent Protein Kinases • Protein Kinase C • Requires calcium, diacylglycerol, and phospholipid for activity • Diacylglycerol generated by the action of phospholipase C • Activated by phorbol esters (tumor promoters) • Many isozymes • Calcium-calmodulin-Dependent Protein Kinases • CAM Kinases I, II, III, IV • Several other protein kinases activated by calmodulin including myosin-light chain kinase, phosphorylase kinase, some isoforms of adenylyl cyclase and some isoforms of phosphodiesterase

  32. Calcium-Dependent PKs

  33. Hormones and Phospholipase C • Heterotrimeric Gq activates PLC No need to memorize any of these

  34. Hydrolysis of PIP2 • Two second messengers are generated • DAG activates PKC • IP3 leads to a rise in cytosolic Ca2+ • This activates PKC and CaM Kinases • Fig. 12-6

  35. Protein Kinase C Family • PKC • C refers to calcium • DAG and phospholipid were also described as necessary for the activation of this enzyme • There are many isozymes that are products of different genes • It is paradoxical to have PKCs that are independent of Ca2+ and DAG

  36. Selected Protein Kinase C Substrates • Glycogen synthase (at least two sites, inactivates) • PDGF receptor • EGF receptor • Insulin receptor • Transferrin receptor • Ribosomal protein S6 • Raf • No need to memorize any of these

  37. Calcium-calmodulin Dependent Protein Kinases • CAM Kinase I: synapsin I and II • CAM Kinase II • CAM Kinase II (autophosphorylation) • Tyrosine hydroxylase (rate-limiting for catecholamine biosynthesis) • CREB transcription factor • Many others • CAM Kinase III: Elongation factor II of protein synthesis (This is a dedicated protein kinase) • CAM Kinase IV • Found in the nucleus • Also phosphorylates the CREB transcription factor • Many others

  38. Receptor Protein-Ser/Thr LigandsTransforming Growth Factor-β Ligands • This family has diverse functions (that you don’t need to remember) • BMP2 subfamily: BMP2 and BMP4, chondrogenesis and other developmental functions • BMP5 subfamily: BMP5,6,7,and 8, development of nearly all organs • BMP3/osteogenin subfamily: bone formation • Activin subfamily: erythroid cell differentiation • TGF-β1, 2, and 3: control of proliferation and differentiation; production of the extracellular matrix • There are about 15 BMPs • They are synthesized as integral membrane proteins and the BMP is cleaved extracellularly in a regulated fashion • Proteins contain about 450 aa; BMPs are about 110 residues • BMP-2 and BMP-4 are expressed by human adult pulp tissue • BMP 1 is a C-terminal procollagen protease

  39. Smads • Transforming growth factor beta ligands including the BMPs activate receptor protein Ser/Thr kinases • Smad transcription factors are phosphorylated and activated • Smads enter the nucleus to bring about a response • These human proteins are homologous to Drosophila proteins called Sma or Mad, thus smad • Smads play a role in normal cell growth, cell division, and apoptosis (programmed cell death)

  40. TGF-β: A Model for the Smad Signaling Pathway

  41. Protein-Tyrosine Kinases • Receptor • Insulin • Epidermal Growth Factor • Platelet Derived Growth Factor • Non-receptor • Src protein kinase • Abl and Bcr-Abl • Jak (Janus kinase [two catalytic regions]) or whimsically, just another kinase

  42. Human Protein-Tyrosine Kinases from the Human Genome Project • The protein-tyrosine kinases are a large multigene family with particular relevance to many human diseases, including cance • A search of the human genome for tyrosine kinase coding elements identified several novel genes and enabled the creation of a nonredundant catalog of tyrosine kinase genes • Ninety unique kinase genes can be identified in the human genome, along with five pseudogenes • Of the 90 tyrosine kinases, 58 are receptor type, distributed into 20 subfamilies • The 32 nonreceptor tyrosine kinases can be placed in 10 subfamilies

  43. Epidermal Growth Factor Receptor Family • In the 1950s-60s Stanley Cohen discovered a factor present in crude submaxillary preparations that induced precocious tooth development in newborn mice that he called epidermal growth factor • He purified EGF, determined its sequence, studied its binding to the EGF receptor, showed that a single molecule contained EGF binding and protein kinase activity • He demonstrated that the EGF receptor was a protein-tyrosine kinase, the first to be described (1980) • He also showed that EGF and receptor are taken up by cells and are degraded in lysosomes (many receptors undergo this fate)

  44. EGF Growth Factor and Receptor Family • Null mutants of any family member are embryonic lethal • Important in development • Implicated in many cancers

  45. ErbB and Malignancies • Head and neck squamous cell carcinomas (>90% associated with ErbB overexpression) • 27,000 new cases in the US per year • Bladder • Breast • Kidney • Non-small cell lung • Prostate cancers • Many other solid tumors

  46. CA of the Tongue • Early squamous cell carcinoma of the tongue • Malignant neoplasms of the oral cavity account for 3-5% of all malignancies • 50% involve the tongue (lateral border and ventral surface most common) • Then floor of the mouth > gingiva > alveolar mucosa > buccal mucosa > palate • Squamous cell carcinomas account for >90% of all malignancies of the oral cavity • Men/woman = 2/1 • Usually more than 40 years of age • Under diagnosed; more than 50% have metastasized at the time of Dx

  47. Receptor Activation • Ligand binds, dimers form, transphosphorylation occurs, and the receptor is activated • Homodimers: ErbB1/ErbB1, etc. • Heterodimers: ERbB1/ErbB2 (common in breast cancer) • One of the dimers phosphorylates the other, and the other dimer phosphorylates the one

  48. How Does the Growth Factor Activate the Receptor?

  49. Monoclonal Abs in the Rx of Cancer • Mabs are directed toward the ectodomain of the ErbB2/HER2 receptor • Herceptin • 20-30% of all human breast cancers overexpress ErbB2, or HER2 (Human Epidermal growth factor Receptor) • These tumors can be treated with Herceptin • It targets domain IV of ErbB2 • Erbitux • Treatment of colorectal cancer that has spread • In combination with irenotecan (a DNA topoisomerase I inhibitor) • From ImClone (The Martha Stewart case) • Approved by US FDA in February 2004

  50. Structure of the EGF Receptor Protein-Tyrosine Kinase Domain • Open activation loop is active (blue or green) • Compact activation loop is inactive (magenta) • This is an important regulatory concept • Blue: EGF unphosphorylated • Green: IRK phosphorylated • Magenta: IRK unphosphorylated

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