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  1. 1. 3.22.2010<br /><ul><li>An overview of Calcium Regulation Cells
  2. 2. 10^-7M of Ca 2+ in cytoplasm
  3. 3. 10^-3 of Ca2+ in ER
  4. 4. IP3 receptors on smooth ER
  5. 5. Ryanodine receptors
  6. 6. Bound by ryanodine, and calcium goes through channels to the cytoplasm
  7. 7. Work like IP3 receptors
  8. 8. On the plasma membrane there are many Ca2+ channels/receptors
  9. 9. Mechanical receptors – stretching membrane
  10. 10. Voltage gated
  11. 11. Seen when you generate an action potentials (open to increased positive charge on the inside of the PM)
  12. 12. Response to increase positive charge on the inside of the plasma membrane for release of neurotransmitters
  13. 13. Ligand gated calcium channels (L-type calcium channels)
  14. 14. When opened, also allow Ca2+ to come into cytoplasm
  15. 15. Calcium Regulation
  16. 16. ATPase in the ER uses hydrolyzed ATP to bring in calcium into the smooth ER, endergonic
  17. 17. Calcium pumps to go into mitochondria (doesn’t really need ATP)
  18. 18. Calcium exchangers to keep cytosolic concentration low (Na+ in/out Ca2+)
  19. 19. Phospholipase c beta
  20. 20. Linked to Heterotrimeric G protein linked receptors
  21. 21. Activates PLCB and cleaves PIP2 in the membrane
  22. 22. Phospholipase c gamma
  23. 23. links up with enzyme linked receptors aka receptor tyrosine kinases (RTKs)
  24. 24. transmembrane receptors that bind ligands on extracellular side of cell and dimerize to phosphorylate the two transmembrane components of the receptor, and there are lots of protein that recognize the phosphorylated sites on the receptor, one of which is PLCG. PLCG binds to the phosphorylated site on receptor and cleaves PIP2, increasing IP3 and DAG signaling (IP3 to increase Ca2+)
  25. 25. The Structure and Activation of a Receptor Tyrosine Kinase
  26. 26. Extracellular part of receptor binds ligand (usually growth factors or cytokines)
  27. 27. RTK Ligands
  28. 28. Cytokines
  29. 29. interleukins
  30. 30. Growth factors
  31. 31. EGF – epithelial growth factor
  32. 32. FGF – fibroblast growth factor
  33. 33. NGF – Nerve growth factor
  34. 34. Neurotrophins
  35. 35. All bind receptor tyrosine kinases (RTKs)
  36. 36. When ligands bind to binding site, the binding sites initiates a conformational change that exposes amino acids in the PM that brings two components of receptor together.
  37. 37. Transmembrane region has attracting amino acids
  38. 38. Brings two monomers together to form a dimer
  39. 39. Juxtamembrane region helps hold receptors together and help attract the monomers to form the dimer (along with transmembrane region)
  40. 40. Cytoplasmic domain has the functional domain where tyrosines are phosphorylated by the neighboring cytosolic tail, so it [cytosolic region] has both the ability to phosphorylate and the sequence to be phosphorylated by its neighbor.
  41. 41. When brought close by amino acids that attract, the phosphorylating domains of cytosolic tail can reach across and phosphorylate the tyrosines on neighboring side of the receptors
  42. 42. Called Autophosphorylation, or cross phosphorylation, because they phosphorylate a similar protein, fundamentally exactly the same.
  43. 43. The pattern of phosphorylation on these receptors determines what types of downstream signaling happens after the receptor is activated.
  44. 44. Each tyrosine can be a binding site for binding of different types of messengers in the cell, which will lead to different pathways being activated.
  45. 45. The specificity in terms of activating specific pathways is determined by what amino acids are on either side of the tyrosine and how they are recognized by downstream signaling proteins
  46. 46. Parts of the RTKs
  47. 47. Extracellular – ligand binding
  48. 48. Trans membrane– used to attract monomers to form the dimers
  49. 49. Juxtamembrane – used to attract monomers to form the dimers
  50. 50. Cytosolic – phosphorylation function
  51. 51. We can call it autophosphorylation because they phosphorylate a similar protein. Both proteins are fundamentally the same. Cross phosphorylate neighboring tyrosines.
  52. 52. The amino acids on either side of the tyrosines can regulate what type of signaling occurs.
  53. 53. A dimer-like signal can also bind to the binding site
  54. 54. All of these intracellular signaling proteins have a domain called an SH2 domain.
  55. 55. Proteins use this SH2 domain to recognize this phosphorylated tyrosine and its amino acids for binding sites for the domain as well as turning on different intracellular signaling pathways
  56. 56. Src Homology domain = SH2.
  57. 57. Src = Sarcomere
  58. 58. Terminology came from tumorigenic cells. People had noticed that there were tumors formed in this tissue and wanted to figure out what was regulating this proliferation of cells. Well, the mutation was in the src homology domain. The mutation resulted in constant RTK signaling, like it was always on. Mutation in SH2 domain always allowed it to activate signaling pathway. Allowed it to activate signaling pathway so the cells continued to divide. SH2 turns on signaling and pathways can regulate cell mitosis.
  59. 59. Proteins with SH2 domains are usually called Protooncogenes – have a mutated form that has been identified as oncogenic, or associated with specific tumors. They are genes that make proteins that when mutated, have an oncogenic form that can induce tumors/cancer.
  60. 60. All of these signaling proteins have an SH2 domain which means they can recognize cerain phosphoryalted tyrosines and turn on their specific intracellular signaling pathway.
  61. 61. RTK signaling continued
  62. 62. Dominant-negative mutations
  63. 63. Any mutation that knocks out or removes function of that protein
  64. 64. Tyrosine kinase domain has been removed and when signaling molecule binds, it cannot autophosphorylate so receptor doesn’t work and there is no signaling beneath it.
  65. 65. Determined to figure out which pathways are activated by the tyrosines
  66. 66. Tyrosine kinase mutation is removed and when signal molecule binds, there is no signaling.
  67. 67. Constitutively activating mutation
  68. 68. Generate receptors that are always on
  69. 69. Receptors that are found in tumorigenic cells and usually responsible for these mutations
  70. 70. Protooncogene -Per2 – major component of some types of breast cancer - Has valine amino acid in transmembrane region in normal form, and within region we have amino acids that help dimerize receptors that are only exposed upon conformationl change.
  71. 71. In the oncogneic mutation, valine is mutated to glutamine (and attract one another and leads to activation of receptor), which causes spontaneous dimerization of the receptor, and act as if growth factor is bound. Recepteors dimierizae and act as if growth factors were bound.
  72. 72. People study these to determine what is the effect of activating these types of receptors.
  73. 73. In the EXS, EGF receptors normally would have to bind EGF. There are certain mutations that remove extracellular region and the absence of extracellular part leads to dimerization and leads to active receptor at all times.
  74. 74. Activation of Receptor Tyrosine Kinase
  75. 75. Linked to activation of small g proteins
  76. 76. MAP Kinase Pathway
  77. 77. Begins with the activation of a small g protein called Ras
  78. 78. Ras links up with RTK Activation and itself is then activated. These small g proteins are kinases themselves and will Phosphoporylate targets on serines and threonine. They are intimately linked to regulation of cell mitiosis and cell differentiation. Remember small g proteins are active when bound to GTP. Guanine Nucleotide Exchange Factors remove GDP and allow GTP to bind, and GAPs enhance andogenous hydrolysis of GTP to leave a GDP for inactivity
  79. 79. Linking to RTKs requires linkers proteins (all of this occurs at plasma membrane)
  80. 80. Ras is linked to inner leaflet of PM and Phosphporylation of the RTK leads to Ras activation indirectly through SH2 protein that can bind the RTK and another protein that acts as a Ras-GEF
  81. 81. An SH2 domain protein links a GEF to Ras at the membrane
  82. 82. Growth factors dimerizes receptor
  83. 83. Receptor undergoes autophosphorylation
  84. 84. A specific phosphorylated tyrosine is recognized by the SH2 domain on the adapter protein (Grb-2 for Ras pathway)
  85. 85. Binding of Grb-2 to Phosphorylated tyrosine releases or activates a region on protein that can be recognized by the GEF
  86. 86. The Ras GEF is called SOS (sauce) – ‘son of sevenless’ because the protein was indentified in drosophila.
  87. 87. Drosophila have eyes that have 8 omatidia, but when mutated, 7, a mutation on SOS protein because the eye cells don’t differentiate correctly.
  88. 88. SOS exchanges GDP for GTP to activate Ras
  89. 89. Once Ras is activated
  90. 90. Sets up a series of phosphorylation events that can regulate gene transcription, also cytoskeletal dynamic, or transport of proteins in cells. Ras is a highly diverse pathway.
  91. 91. Downstream targets are also phosphorylated etc etc
  92. 92. MAP Kinase Kinase Kinase phosphorylates MAP kinase Kinase
  93. 93. MAP Kinase Kinase phosphorylates MAP Kinase
  94. 94. Or Ras Raf MAPK Erk1/2 (MAP Kinase)
  95. 95. Erk can go to nucleus and regulate gene transcription by phosphorylating target proteins in the nucleus that regulate gene transcription
  96. 96. Numerous targets for Ras pathway
  97. 97. -------------------------------------------------------------------------------------------------------
  98. 98. Grb-2 adapter proteins that has SH2 domain, binds to Phosphorylated tyrosine
  99. 99. Recruits SOS to membrane
  100. 100. SOS exchanges GDP for a GTP to activate Ras
  101. 101. Ras sets up a series of phosphorylation events that can regulate gene transcription or cytoskeleton dynamics or transport of proteins within the cell
  102. 102. Leads to series of Phosphorylation events that phosphorylates downstream targets
  103. 103. Ras phosphporylates MAP kinase kinase kinase (RAF)
  104. 104. Which phosphorylates MAP kinase kinase (MAP kinase)
  105. 105. MAP kinase (erk)
  106. 106. Or Ras Raf MAPK Erk1/2 (MAP Kinase)
  107. 107. Erk can go to nucleus and regulate gene transcitpion by phosphorylating target proteins in the nucleus that then regulate gene transcription
  108. 108. -------------------------------------------------------------------------------------------------------
  109. 109. Pathway uses ATP hydrolysis for phosphorylation event
  110. 110. Each kinase is a serine/threonine kinase
  111. 111. EGF growth factor binds and dimerizes EGF receptor
  112. 112. EGF receptor then undergoes autophosphorylation
  113. 113. The autophosphorylation recruits Grb-2 as well as SOS to the membrane (Ras-GEF) [this must ALL occur near the membrane because Ras is bound the the PM].
  114. 114. SOS will take out the GDP (near Ras) and allow GTP to bind. When Ras is activated, that Ras will bein to Pi the MAP Kinase Kinase Kinase, which then Pi MAP Kinase Kinase, then Pi MAP Kinase,
  115. 115. In Pi’ed form, MAP Kinase/Erk has a NLS that will allow it to be carried to the nucleus and turn on transcription factors like AP-1
  116. 116. What is AP-1?
  117. 117. A complex made of two proteins (which are individual in the nucleus)
  118. 118. Jun
  119. 119. Phosphorylated by MAP Kinase/Erk first, and then it recruits Fos after Fos is brought to Jun
  120. 120. Fos
  121. 121. Pi’ed after brought to Jun.
  122. 122. Once Jun and Fos are brought together, they function as a transcription factor, which will upregulate gene transcription (for cell division and channels etc)
  123. 123. What is on the other side of the pathway?
  124. 124. Activation of PLCG!!!
  125. 125. A RTK activates phospholipase signaling
  126. 126. Because has PLCG has a different SH2 domain that binds to a different Pi’ed tyrosine on receptor
  127. 127. PLCG cleaves PIP2 in membrane
  128. 128. Generating DAG and IP3 and binds IP3 receptor to release calcium, which will bind calcium binding proteins to other pathways or Ca2+ can migrate to DAG and Phosphotidle serene to activate PKC
  129. 129. Pathway cross talk
  130. 130. Activated G protein
  131. 131. Activation of PKA because that G protein activates HTGP that activates Adenylyl cyclase
  132. 132. PKA will Pi target proteins and involved in gene transcription
  133. 133. Same or diff. g protein can activate PLCB leading to IP3 and DAG signaling
  134. 134. IP3 leads to PKC activation or calmodulin Cam Kinase activation
  135. 135. MAP kinase pathway regulates gene transcription (just look at the chart on the second to last slide)
  136. 136. At level of substrate level phosphorylation:
  137. 137. Transmembrane proteins may activate same protein or may activate separate proteins that the come together later.
  138. 138. Two products from two different pathways will come together and continue a signal relay.
  139. 139. ***Cell Cycle Regulation***
  140. 140. Four discrete phases of the cell cycle
  141. 141. Prophase
  142. 142. Prometaphase
  143. 143. Metaphase
  144. 144. Anaphase
  145. 145. Telophase and cytokinesis
  146. 146. Interphase
  147. 147. G1 and G2
  148. 148. Makes sure DNA is replicated correctly and they both contain a checkpoint.
  149. 149. S Phase