Lecture 15 &16 Review Guide

Which enzymes regulate the phosphorylation status of a protein?

Lecture 15  

o [Writing Assignment] Phosphorylation plays a major role in the regulation of signal  transduction. What does phosphorylation do in the context of signaling?

Phosphorylation can activate or deactivate things. It regulates signaling pathways.  o Which enzymes regulate the phosphorylation status of a protein?

The enzymes that regulate phosphorylation include kinase (add phosphate groups) and  phosphatases (remove phosphate groups).  

o How does the activity of these enzymes affect the outcome of a signaling pathway?

Phosphorylation creates binding sites. If you recruit something that promotes signaling there is  activation. If you recruit something that inhibits, the signaling pathway will be shut off.  

How does the activity of these enzymes affect the outcome of a signaling pathway?

o p53 is a transcription factor that is activated through phosphorylation in response to  DNA damage. Active p53 halts the cell cycle. Predict the outcome of a mutation in the  phosphorylation site on p53. If you want to learn more check out How accurate is the humanistic perspective?

p53 cannot be phosphorylated in response to DNA damage. It cannot be activated, and it  cannot inhibit cell cycle, which leads to proliferation of this damaged state, and cancer can be  the result.  

∙ Describe the MAPK signaling pathway?

There are a lot of different receptors and a lot of then will activate Ras which will often result in  activation in MAP Kinases. Map Kinases are a series of kinases that are held together by a  scaffold protein and function in helping to relay the signal from the receptor to the effector  protein through phosphorylation.  

Describe the mapk signaling pathway?

∙ Describe Ras activation? How is it quickly reversed?

Ras activation is brief and transient – quickly reversed by the activity of phosphatases. Ras is  involved in spreading the signal to multiple pathways. If you wanted to turn off signaling you  could add a phosphatase to dephosphorylate the receptor to lose the GEF. You’ll get shut off  and hydrolysis of GTP and shut off of Ras.  

∙ What is FRET? What is it used for?

FRET is fluorescence resonance energy transfer and it is used to study RAS activation. Different  fluorescence is activated by different wave lengths. You can use one fluorescence to activate  another. If two fluorescence molecules are close together then one energy can activate the  other and you can get a color change. FRET is a way to asses if Ras is active or not.  

In this experiment they have a signaling pathway that is responsive to epidermal growth factor  (EGF) which will bind to EGF receptor, and then you’ll have a pathway that requires Ras. You  can tell if Ras is activated or not based on the amount of red. They added EGF and over time  there is an increase in activated Ras by looking for the red, and over time it quickly comes  down. This is through the activity of phosphatases that quickly shuts things off. FRET allows you  to measure the proximity of two molecules based on color.  Don't forget about the age old question of Define demand curve.

Signaling gets turned on for a purpose but it needs to be turned off. Growth factors turned on  forever is really bad. The role of phosphatases is very important.  

∙ What do Rho GTPases couple?  

Rho is another GTP binding protein (like an on/off switch for something). Rho GTPases couple  cell surface receptors to the cytoskeleton.  

∙ Describe how signaling affects cell shape. What is an example?

They regulate actin polymerization and regulate cell shape and motility. The Eph receptor is an  example.  

∙ What affects cell shape? Describe the mechanism.  

Activation of the Rho family of GTPases by signaling through the Eph receptor affects cell shape.  If you want to learn more check out What is mhc? why is needed in the immune system?

Motor neurons need to grow towards the muscle to interact, and certain motor neurons need  to go in the opposite direction. There is regulation to make sure there isn’t a lot of motor  neurons growing in the same direction. There is interaction between two receptors on two  different cells. The Eph receptor binds to ephrin A1 and when binding occurs, there is  phosphorylation and recruitment of Rho-GEF. The Rho-GEF will activate some GTP binding  protein (Rho-A).

∙ Describe growth cone collapse in motor neurons.

Contraction of the cell causes the growth cone (the tip of the cell that is growing towards the  other cell) of the cell to collapse. It is a way of saying “no, don’t grow here.”

∙ What does Rho A do?

Rho A stimulates myosin dependent contraction of actin cytoskeleton. There is actin  polymerization and there is myosin-mediated-filament-contraction.  

∙ Is guidance of axon tips to muscle target signaling negative or positive regulation? Guidance of axon tips to muscle target signaling is negative regulation.  If you want to learn more check out What is an inventory system where companies manufacture or purchase goods only when needed for use?

∙ What happens in the absence of signal ephexin?

In absence of signal ephexin activates all Rho GTPases equally = forward movement of axon tip  towards muscle target.  If you want to learn more check out What idea is discussed in behavioral allocation view?

When there is no signaling, there will be activation of all different types of Rho-GTPases. The  motor neuron grows. There is a growth cone extension because all of the Rho-GTPases are  going to work together for this growth process of the cell.  

∙ What happens in the presence of a signal?

In the presence of a signal = preferenced for Rho-A activation, contraction only = growth cone  collapse.

When you get signaling, everything is shut off except for Rho-A and this causes contractile actin,  and this causes the growth cone to collapse. It is negative regulation because it is saying “no  neuron, don’t grow towards me.” Don't forget about the age old question of What are the two products of photosynthesis?

∙ What do PI 3-kinase phosphorylates inositol phospholipids do? What can activate it?  

PI 3-Kinase phosphorylates inositol phospholipids creating docking sites for intracellular  signaling proteins. This can be activated by RTSs, GPCRs, or Ras.  

PI 3-Kinase is a kinase that can be activated by many different receptors. It phosphorylates  inositol phospholipids in the plasma membrane and creates docking sites for other proteins. It  activates another signaling pathway. PI 3-Kinase regulates different pathways.  

∙ What is the outcome of a mutated PTEN?  

PTEN is commonly mutated in cancer. Phosphorylation ???? binding sites ???? signaling pathways  associated with cell growth and survival = PTEN shuts this off

If PTEN was mutated, the pathway will always be turned on because you will never have the  removal for phosphate groups. PI 3-Kinase adds all sorts of phosphate groups there, and PTEN  removes them. Those phosphate groups are binding sites for different proteins associated with  signaling

∙ What is PI3-K involved in?  

PI 3-Kinase is involved in cell growth and survival  

∙ What does signaling through PI3K-Akt pathway promote?  

Signaling through PI3K-At pathway promotes growth and survival.  

There is phosphorylation and dissociation of an AKt protein, a kinase. It is active and  phosphorylates things. One of the targets that it phosphorylates is a Bad protein. Bad normally  binds to and keeps the inhibitor of apoptosis inactive. When Bad is phosphorylated it goes and  binds to something else and now there is an inhibitor of apoptosis that is active. It goes on and  inhibits apoptosis. Apoptosis, from the perspective of the cancer patient, is good because you  want to kill the cancer cells.  

∙ What happens in cancer and which genes might be affected in cancer?

Cancer is a dysregulated cell cycle (cell cycle that keeps happening) combined with inhibition of  apoptosis. Genes effected in cancer include PI3-Kinase, PTEN (takes off phosphate groups), AKt,  Mtorr  

∙ Describe crosstalk.

Crosstalk between pathways activated by different kinds of receptors. Things can be very  complicated.

∙ Describe tyrosine-kinase-associated receptors.

Rather than having their own intrinsic kinase activity like RTKs, tyrosine kinase associated  receptors interact with and signal through cytoplasmic tyrosine kinases – usually memers of the  Src family of tyrosine kinases.  

All are kinases that contain SH2, Sh3 domains and bind to cytoplasmic domain of receptors.  Receptors that bind to a kinase, they do not have a kinase domain. They usually interact with  members of the Src family.  

∙ Describe SH2 and SH3 domains.

SH2-bind phosphor tyrosine

SH3 – bind to proline rich sequence in other protein P-X-X-P (a protein-protein interaction)  

∙ How is the Jak-Stat pathway an example of signaling through a tyrosine kinase  associated receptor?

When there is binding of the signaling molecule and receptors dimerize and come close  together there is activation of the kinases (TYK2 & Jak1). They phosphorylate each other and  the cytoplasmic tail, creating binding sites for the STAT1&STAT2 proteins. They phosphorylate  the STAT1&STAT2 proteins which allows them to release from the receptor and dimerize. They  can go into the nucleus and activate expression with genes associated with the antiviral  response.  

∙ How is signaling turned on and off?  

Signaling is turned on by alpha-interferon secreted by some other cell, bound to a receptor. To  turn this off you could pull it into a multivesicular body (endocytosis). You can also remove  phosphate groups if you have a phosphatase.  

∙ Why would you want to turn it off?

You would want to turn it off because you don’t need transcription going on all the time. You  don’t need transcription of anti-viral genes when the virus is gone.  

∙ What do tyrosine phosphatases do?

Tyrosine phosphatases modify and/or turn signals off.  

∙ Describe tyrosine phosphatases.

They can be cytoplasmic or plasma membrane bound. The role of plasma membrane bound is  unclear.

∙ How would a phosphatase modify a signal?

They could modify a signal by removing a phosphate group, which could change some of the  signaling cascade. Modifying the activity because there could be multiple phosphorylation sites  and maybe just one is removed, and that’s changing the activity somehow.  

∙ How would it turn off a signal?

∙ Describe Smad Dependent TGF-B signaling. What is it involved in?  

Smad dependent TGF-B signaling is another example of signaling through a receptor-kinase (has  kinase domain in receptor). It has a serine/threonine kinase domain and is involved in  development, tissue repair, and immune regulation. TGF-B binds to the receptor and you get  activation, dimerization of the receptors, phosphorylation, activation, and binding sites for  recruitment of Smad.

∙ Smad4 is an example of a latent gene regulatory protein – what is this?

Latent gene regulatory proteins are a gene regulatory gene protein that is dormant/inactive.  The gene regulatory protein is in the cytoplasm but it’s in an inactive form. It cannot go into the  nucleus and it cannot activate gene expression.

o [Practice Problem] Phosphorylation of Smad induces interaction with Smad 4 and  translocation to the nucleus. What assumptions could you make about Smad 2/3  and/or 4 in terms of nuclear localization?

Together the Smads could be creating a NLS sequence. Smad 2/3 could not be located in the  nucleus, and a reason for that would be that maybe it needed to be bound to Smad 4 in order  for there to be a NLS made up of the two of them.  

Maybe 2/3 don’t have a NLS and when it dimerizes with Smad 4 (Smad 4 actually has a NLS that  is exposed) and then maybe it could go in the nucleus.  

Smad 2/3 could only be phosphorylated in the cytosol, because that’s where their kinase is.  Smad 4 could be allowing it to go in the nucleus.

Maybe dimerization creates some sort of shape that allows it to go into the nuclear pore.  

It definitely has to do with a NLS. Smad 2, 3, and 4 are all folded until they dimerize. Any NLS  can be obscured, and there is a massive conformational change when it is dimerized. This  allows a NLS to be exposed on Smad 2, 3, or 4.  

∙ Describe signaling pathways that depend on regulated degradation/proteolysis.  

Signaling pathways that depend on regulated degradation/proteolysis to control the activity of  latent gene regulatory proteins. – A gene regulatory protein that is there but isn’t active until  something comes and activates it.  

∙ Describe Notch.

Notch signaling plays a role in development, cell fate, and pattern formation in the  development of tissues.  

∙ Describe Wnt.

Development (recitation paper)  

∙ Describe NFkB.

Stress response, inflammatory response, innate immune response

∙ What does Notch signaling depend on?  

Notch signaling depends on the regulated cleavage of the Notch cytoplasmic tail.  ∙ Describe the pathway in Notch.  

Notch signaling is a mechanism for communication between adjacent cells and involves lateral  inhibition. An example is epithelial cells that need to differentiate and interact. There is  interaction between delta (on the neighboring cell, the signaling cell) and notch (on the cell that  will respond). Notch signaling can promote or inhibit a developmental program.

Travels to nucleus to bind to a DNA binding protein switching a transcriptional repressor to a  transcriptional activator.  

There is a Notch receptor. Any receptor is going to be translated in the ER and it will go through  the secretory pathway. One of the processing steps is cleavage in the Golgi. There is a signaling  cell and a responding cell. There is two different receptors. Delta is on the signaling cell (the  signaling molecule) and Notch on the cell that is going to respond. There will be binding of Delta  and Notch, and then there is activation of protease that ends up cleaving Notch. The  cytoplasmic tail dissociates from the receptor, it is the effector protein, the transcription factor.  It migrates to the nucleus and interacts with different gene regulatory proteins, and there is  activation of gene expression.  

Latent gene regulatory protein that is normally part of the receptor, it is normally the  cytoplasmic tail of the receptor. When you have receptor binding to the signaling receptor from  the other cell, you’ll get cleavage of the cytoplasmic tail, and then it goes to the nucleus and  activates gene expression.  

∙ How is Notch similar to UPR?  

Notch signaling is similar to UPR because one of the sensors. Also in both mechanisms the  cytoplasmic tail got cleaved. In UPR, after it sensed the misfolded the proteins in the ER, a  protease was activated, cleaved the cytoplasmic tail, and the cytoplasmic tail traveled to the  nucleus to activate gene expression. The response was to make more proteins. In notch, it is  involved with development and differentiation in cells.  

∙ What does Wnt signaling depend on?  

Wnt-B-catenin signaling depends on inhibition of the degradation of B-catenin.  

Without Wnt, B-catenin is constantly being degraded. It’s interacting with APC destruction  complex which is keeping GSK3 close by and active, and is phosphorylating B-catenin.

Phosphorylated B-catenin is recognized by the ubiquitin proteasome system, it is ubiquinated  and degraded. Signaling is off because the gene regulatory protein is being degraded. Constant  degradation is happening here. When you have signaling through Wnt binding to the receptor  then the whole complex is recruited to the receptor, goes through endocytosis and it is all  sequestered in multivesicular bodies. The GSK3 protein is the kinase that targets B-catenin to  be degraded and is sequestered in multivesicular bodies, which allows B-catenin to be stable  and go activate gene expression.  

∙ How is B-catenin stabilized?  

B-catenin is stabilized through recruitment of Wnt signaling complexes into MVB.  

When Wnt binds there is endocytosis of the whole pathway including GSK3, and it goes to  MVBs and this allows B-catenin to be stable.  

∙ What does NFkB signaling depend on? What is IkB?  

NFkB signaling depends on regulated proteolysis of IkB, an inhibition. IkB is being degraded.  When you get signaling through the receptor you get activation of this IKK complex which  contains a protein (a kinase called NEMO) and the kinase complex phosphorylates IkB and then  it gets ubiquinated and degraded, and that’s what exposes the NLS on NFkB so it can go into the  nucleus and activate gene expression.  

NFkB is another example of a latent gene regulatory protein (NFkB) regulated by regulated  proteolysis (of IkB, the inhibitor).

∙ When does signaling need to be turned off?  

∙ Describe receptor endocytosis.  

∙ What does B-arrestin do?  

∙ What family of proteins might b-arrestin have similarity to?

∙ What can happen to receptors?  

∙ Describe sequestration in a multivesicular endosome.  

∙ What can receptor phosphorylation do? What happens?

∙ “Deactivating the G-protein through GTP hydrolysis – induced by binding the target  protein or interacting with an RGS protein which acts as a GAP”

∙ Describe the inhibition of the JAK-STAT pathway. What are the two mechanisms? Lecture 16

o [Writing Assignment] What is a latent gene regulatory protein?  

A latent gene regulatory protein is a protein in the cytoplasm that is inactive in the form that it  is in and gets activated somehow.

o How are these genes regulated (turned on and off)?

Genes are regulated through signal transduction and proteolysis of a regulatory protein.  Phosphorylation might result in a conformational change. They could also be regulated through  some kind of competitive inhibition and binding to another protein (maybe an inhibitor)  

o Give 3 examples of latent gene regulatory proteins and explain how they are  regulated.  

B-catenin ???? [Pathway = Wnt] B-catenin is regulated through endocytosis of GSK3. GSK3  endocytosis is what controls degradation of B-catenin. B-catenin is usually being degraded, and  activation of the Wnt pathway causes it to be stabilized. Regulating stability.  

Cytoplasmic Tail of Notch ???? [Pathway = Notch] the cytoplasmic tail is regulated through  cleavage. Once Delta binds Notch and the pathway is stimulated, you get cleavage of the  cytoplasmic tail of Notch and that is what turns it on and allows it to go to the nucleus.  

NFkB ???? [Pathway = NFkB] degradation of the inhibitor, NFkB is the latent gene regulatory  protein and it is regulated through controlling the stability of the inhibitor  

o Consider how these pathways might become dysregulated in ways that would  promote disease/pathology.

Wnt ???? if there is an issue in forming multivesicular bodies GSK3 will be active. This effects  signaling because B-catenin will be degraded and there will be no gene expression. MVBs are  also very important for a lot of things

No degradation of B-catenin = constant signaling, constant gene expression  APC ???? issue with the destruction complex  

Constitutively active receptors ???? keep signaling on  

NFkB ???? if you don’t have the kinase phosphorylating IkB then you’ll never activate gene  expression because IkB won’t be degraded which would keep the pathway off, which is bad if  you want any kind of immune response. Mutation in the inhibitor protein so it cannot bind to  NFkB, so NFkB will always be on and that is also bad. In both cases you’re losing regulation.  

∙ When does signaling need to be turned off?  

Signaling needs to turn off once the goal of the signaling pathway is accomplished. Signaling  that is unregulated is bad.

∙ What is the major mechanism #1?

The first mechanism is inhibiting signaling at the receptor level.

∙ What are some ways you can inhibit a receptor?

Endocytosis ???? Downregulation through receptor mediated endocytosis and it goes into a MVB Endocytosis & Degradation ???? you can have endocytosis that eventually will be recycling, and  then you can have endocytosis going to the lysosome – both are downregulation that are  pulling the receptor off the cell surface

Inactivation ???? through interaction of an inhibitor with the cytoplasmic tail – blocking  intracellular signaling molecule

Modification of the receptor ???? a phosphorylation that is inhibitory can be useful in recruiting  an inhibitor or you can have a signaling protein that gets shut off (ex: Ras turns off signaling)  Negative feedback ???? activation of the pathway will result in the expression of a protein that is  an inhibitor  

∙ What is a mechanism for desensitization?

Receptor endocytosis is a mechanism for desensitization

∙ What does B-arrestin do?  

B-arrestin binds receptors and incorporates them into clathrin coated pits.  ∙ What family of proteins might b-arrestin have similarity to?

B-arrestin is acting like adaptin. Adaptin is a family of proteins that interacts with the  cytoplasmic tail of a receptor and clathrin, and recruits the protein into a clathrin coated pit for  endocytosis  

∙ What can happen to receptors?  

Receptors can be endocytosed and recycled or sent to the lysosome. The receptor can go into a  recycling endosome and back to the cell surface, or it goes to the lysosome and is degraded.  

∙ Describe sequestration in a multivesicular endosome.  

Sequestration in a multivesicular endosome (MVE/MVB) that recycles or fuses with the  lysosome. The MVE is so you can hide the cytoplasmic tail that is interacting with signaling  molecules.  

∙ What can receptor phosphorylation do? What happens?

Receptor phosphorylation can inhibit signal transduction. The receptor is phosphorylated with  cAMP dependent protein kinase, activity increases as levels of cAMP increase in response to  epinephrine.  

You can get phosphorylation of the receptor by a kinase that is activated by the signaling  protein (this is similar to arrestin and desensitization).  

∙ How are G-Proteins regulated?

Regulation is done through GAPs and GEFs.  

Activation of a GPCR through ligand binding, kicks off GDP, allowing GTP to bind  Deactivation through GTP hydrolysis  

∙ Describe deactivation of G-proteins

“Deactivating the G-protein through GTP hydrolysis – induced by binding the target protein or  interacting with an RGS protein which acts as a GAP”

Activated G-protein will activate another protein. The GPCR will actually activate this GAP  activity which results in GTP hydrolysis, loss of the phosphate, and this turns off the GPCR

∙ Describe the inhibition of the JAK-STAT pathway. What are the two mechanisms?

Major mechanism #1 ???? inhibiting receptor function somehow

Major mechanism #2 ???? inducing the degradation or inactivating of components of the  signaling pathway

Inhibition of the JAK-STAT pathway through inactivation of the receptor associated kinase  through two mechanisms.  

∙ What are some ways things can be inhibited?  

Inhibition of (Jak) kinase activity

∙ What are two ways things could be turned off?

Inhibition of (Jak) kinase activity

Actual degradation of kinases  

Induce degradation or inactivation of components of the signaling pathway Targeting components of the signaling pathway (JAK here) for proteasomal degradation – this  will shut off signaling

∙ How is signaling turned off?  

Signaling is turned off when phosphatases remove phosphate groups. If a receptor or a  signaling molecule in the middle of the pathway, or even the latent gene regulatory protein is  phosphorylated to activate it – removal of phosphatases can shut it off.  

∙ Give a wrap up on cell communication:

o GPCRs signal through G proteins – regulate signaling through affecting levels of  small mediators such as cAMP and Ca++ (second messengers)  

o RTKs (receptor kinases) have intrinsic kinase activity – activate other kinases  through phosphorylation to relay signal to nucleus

o Kinase associates receptors – similar to RTK but associate with kinase rather than  have own kinase domain (bind to a kinase)  

o Signaling through regulated proteolysis – protein cleavage or degradation  liberates a transcription factor, or inhibition of degradation stabilizes an  

transcription factor

∙ Signal transduction thoughts and concepts:  

o What is the purpose? ???? alter/control/regulate gene expression, motility,  affecting cell shape, cell communication, cell development (Notch and Delta, and  Eph receptor), proliferation (cell division), metabolism – signal transduction  regulates everything  

o How is it a regulated process? ???? ligands activate things, things are turned on  and off

o Why is it a regulated process? ???? you don’t want to have all signaling pathways  always active, you don’t want overexpression of genes when they’re not needed,  you don’t want cells dividing when they shouldn’t be dividing  

o What would be the outcome if it became an unregulated process? ???? cancer or  disease

o How could signaling become unregulated?