3.22.2010

1. An overview of Calcium Regulation Cellsa. 10^-7M of Ca 2+ in cytoplasmb. 10^-3 of Ca2+ in ERc. IP3 receptors on smooth ERd. Ryanodine receptors

i. Bound by ryanodine, and calcium goes through channels to the cytoplasmii. Work like IP3 receptors

e. On the plasma membrane there are many Ca2+ channels/receptorsi. Mechanical receptors – stretching membrane

ii. Voltage gated1. Seen when you generate an action potentials (open to increased

positive charge on the inside of the PM)2. Response to increase positive charge on the inside of the plasma

membrane for release of neurotransmittersiii. Ligand gated calcium channels (L-type calcium channels)

1. When opened, also allow Ca2+ to come into cytoplasmf. Calcium Regulation

i. ATPase in the ER uses hydrolyzed ATP to bring in calcium into the smooth ER, endergonic

ii. Calcium pumps to go into mitochondria (doesn’t really need ATP)iii. Calcium exchangers to keep cytosolic concentration low (Na+ in/out

Ca2+)g. Phospholipase c beta

i. Linked to Heterotrimeric G protein linked receptors 1. Activates PLCB and cleaves PIP2 in the membrane

h. Phospholipase c gamma i. links up with enzyme linked receptors aka receptor tyrosine kinases

(RTKs) 1. 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+)

2. The Structure and Activation of a Receptor Tyrosine Kinasea. Extracellular part of receptor binds ligand (usually growth factors or cytokines)

i. RTK Ligands 1. Cytokines

a. interleukins2. Growth factors

a. EGF – epithelial growth factorb. FGF – fibroblast growth factorc. NGF – Nerve growth factor

i. Neurotrophinsd. All bind receptor tyrosine kinases (RTKs)

ii. 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.

1. Transmembrane region has attracting amino acidsa. Brings two monomers together to form a dimer

iii. Juxtamembrane region helps hold receptors together and help attract the monomers to form the dimer (along with transmembrane region)

iv. 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.

1. 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

a. Called Autophosphorylation, or cross phosphorylation, because they phosphorylate a similar protein, fundamentally exactly the same.

v. The pattern of phosphorylation on these receptors determines what types of downstream signaling happens after the receptor is activated.

1. 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.

2. 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

vi. Parts of the RTKs1. Extracellular – ligand binding2. Trans membrane– used to attract monomers to form the dimers3. Juxtamembrane – used to attract monomers to form the dimers4. Cytosolic – phosphorylation function

a. We can call it autophosphorylation because they phosphorylate a similar protein. Both proteins are fundamentally the same. Cross phosphorylate neighboring tyrosines.

5. The amino acids on either side of the tyrosines can regulate what type of signaling occurs.

vii. A dimer-like signal can also bind to the binding site

3. All of these intracellular signaling proteins have a domain called an SH2 domain. a. 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

i. Src Homology domain = SH2. 1. Src = Sarcomere

2. 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.

ii. 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.

iii. 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.

4. RTK signaling continueda. Dominant-negative mutations

i. Any mutation that knocks out or removes function of that proteinii. 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.

iii. Determined to figure out which pathways are activated by the tyrosinesiv. Tyrosine kinase mutation is removed and when signal molecule binds,

there is no signaling.b. Constitutively activating mutation

i. Generate receptors that are always onii. Receptors that are found in tumorigenic cells and usually responsible for

these mutationsiii. 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.

iv. 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.

1. People study these to determine what is the effect of activating these types of receptors.

v. 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.

5. Activation of Receptor Tyrosine Kinasea. Linked to activation of small g proteins1. MAP Kinase Pathway

a. Begins with the activation of a small g protein called Ras

i. 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

ii. Linking to RTKs requires linkers proteins (all of this occurs at plasma membrane)

iii. 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

1. An SH2 domain protein links a GEF to Ras at the membranea. Growth factors dimerizes receptor

i. Receptor undergoes autophosphorylation1. A specific phosphorylated tyrosine is

recognized by the SH2 domain on the adapter protein (Grb-2 for Ras pathway)

a. Binding of Grb-2 to Phosphorylated tyrosine releases or activates a region on protein that can be recognized by the GEF

b. The Ras GEF is called SOS (sauce) – ‘son of sevenless’ because the protein was indentified in drosophila.

i. Drosophila have eyes that have 8 omatidia, but when mutated, 7, a mutation on SOS protein because the eye cells don’t differentiate correctly.

ii. SOS exchanges GDP for GTP to activate Ras

iv. Once Ras is activated1. 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.

a. Downstream targets are also phosphorylated etc etci. MAP Kinase Kinase Kinase phosphorylates

MAP kinase Kinase

1. MAP Kinase Kinase phosphorylates MAP Kinase

i. Or Ras Raf MAPK Erk1/2 (MAP Kinase)

1. Erk can go to nucleus and regulate gene transcription by phosphorylating target proteins in the nucleus that regulate gene transcription

2. Numerous targets for Ras pathwayb. -------------------------------------------------------------------------------------------------------

i. Grb-2 adapter proteins that has SH2 domain, binds to Phosphorylated tyrosine

1. Recruits SOS to membranea. SOS exchanges GDP for a GTP to activate Ras

i. Ras sets up a series of phosphorylation events that can regulate gene transcription or cytoskeleton dynamics or transport of proteins within the cell

ii. Leads to series of Phosphorylation events that phosphorylates downstream targets

1. Ras phosphporylates MAP kinase kinase kinase (RAF)

a. Which phosphorylates MAP kinase kinase (MAP kinase)

i. MAP kinase (erk)iii. Or Ras Raf MAPK Erk1/2 (MAP Kinase)

1. Erk can go to nucleus and regulate gene transcitpion by phosphorylating target proteins in the nucleus that then regulate gene transcription

c. -------------------------------------------------------------------------------------------------------

i. Pathway uses ATP hydrolysis for phosphorylation eventii. Each kinase is a serine/threonine kinase

iii. EGF growth factor binds and dimerizes EGF receptor1. EGF receptor then undergoes autophosphorylation

a. 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].

i. 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,

1. 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

2. What is AP-1?a. A complex made of two proteins (which are

individual in the nucleus)i. Jun

1. Phosphorylated by MAP Kinase/Erk first, and then it recruits Fos after Fos is brought to Jun

ii. Fos1. Pi’ed after brought to Jun.

b. Once Jun and Fos are brought together, they function as a transcription factor, which will upregulate gene transcription (for cell division and channels etc)

6. What is on the other side of the pathway?a. Activation of PLCG!!!

i. A RTK activates phospholipase signalinga. Because has PLCG has a different SH2 domain that binds to

a different Pi’ed tyrosine on receptor i. PLCG cleaves PIP2 in membrane

1. 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

7. Pathway cross talka. Activated G protein

i. Activation of PKA because that G protein activates HTGP that activates Adenylyl cyclase

1. PKA will Pi target proteins and involved in gene transcription2. Same or diff. g protein can activate PLCB leading to IP3 and DAG

signaling3. IP3 leads to PKC activation or calmodulin Cam Kinase activation4. MAP kinase pathway regulates gene transcription (just look at the

chart on the second to last slide)b. At level of substrate level phosphorylation:

i. Transmembrane proteins may activate same protein or may activate separate proteins that the come together later.

1. Two products from two different pathways will come together and continue a signal relay.

8. ***Cell Cycle Regulation***

9. Four discrete phases of the cell cyclea. Prophaseb. Prometaphasec. Metaphased. Anaphasee. Telophase and cytokinesisf. Interphase

i. G1 and G21. Makes sure DNA is replicated correctly and they both contain a

checkpoint.ii. S Phase