AVPL Problem Set 6 COMPLETE

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Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis lnstructions: ✓ Download this problem set, keep the questions in the document and please type your answers in a color other than black so that your answers are easily discernable. ✓ Complete this entire problem set – give robust, detailed responses – this is practice so use it as such. ✓ If you get stuck or need clarification on any of these questions… just ask! ✓ Upload this document with your answers to the appropriate TurnItIn link in our Moodle page in PDF format ✓ Please be advised that TurnItIn is a plagiarism and similarity checking software o Make sure that you close and put away all sources of information before writing your answers so that you can be sure that your answers are in your own words and to convince yourself that you understand the concept and can explain it yourself. o If your answers are too similar to either someone else’s responses (either from this course or from previous iterations of this course) or from the internet, points will be deducted as the assumption will be made that the work you’re doing is not your own. We'll work on the following problem set questions during Discussion Section this week: Questions 4 and 6. This problem set is worth 15 points: • There will be a 5-point quiz in Discussion Section based on one of the questions worked on in Discussion Section • Once you submit the entire problem set at the end of the week: o 5 points for robust completion of the entire problem set (checking for honest effort but not checking for correctness) o There will be one other question chosen at random and graded for robust answer and correctness worth 5 points Due date: Sunday, April 9 th by 11:59pm

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis General Principles of Cell Signaling and Signal Transduction Question 1: The Concept of Signal Transduction in Cells Cell signaling is the study of how cells communicate with the world outside of their cell membrane. Although we talk about signals being “transduced” across the cell membrane, it is often not the physical signal itself that is moving inside the cell (although sometimes it is). In cells, what is meant by signal transduction? If a physical signal is not moved through the cell membrane (which it often isn’t), then how does the signal (e.g. information) get moved to the inside of the cell and outcome occur? You might use the following resources to think about these concepts – read, synthesize, put away your resources and then answer the question in your own words from your own understanding of the concepts: https://www.khanacademy.org/science/ap-biology/cell-communication-and-cell- cycle/changes-in-signal-transduction-pathways/a/intracellular-signal-transduction https://bio.libretexts.org/Learning_Objects/Worksheets/Biology_Tutorials/Signal_Transd uction https://www.nature.com/scitable/topicpage/cell-signaling-14047077/ Signal transduction is a chain reaction inside the cell that creates an outcome. This chain reaction occurs when a signal outside of the cell interacts with a receptor on the cell. A signal can take many forms, but the main idea is that they activate a receptor located on the outside of the cell by binding to the receptor and changing its shape which then causes it to function in a different way than before. This activated receptor than causes changes in other cells by activating them in some sort of way. This process usually involves phosphates as they usually bind to proteins to activate them. These phosphates are moved around by phosphate kinase which move phosphates around to activate other enzymes and proteins in the cell. The activated proteins can cause many different things to happen depending on the cell, some catalyze other proteins to carry out functions faster while others can activate some sort of repressor and cause other affects within the cell. The outcome of all this is regulation of cellular activities, and it is important for a cell to be able to change its activities based on its environment, that way it knows what it must do to adapt itself based on these signals.

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis Question 2: The Shape, Chemistry, and Size of Signals Signals can come in many shapes and sizes, from nitric oxide (two atoms) to a small protein (lots of atoms). Some signals cannot pass through the membrane and thus need to bind to cell surface receptors. Some signals can pass through the membrane and bind receptors in the cytosol. What chemical characteristics would you expect membrane soluble signals to have and what chemical characteristics would you expect membrane insoluble signals to have? The membrane is made up of tightly packed hydrophobic lipids, the signals that can pass through the membrane tend to be smaller in size and hydrophobic as well. This is because the molecules need to be small enough to get through the gaps within the membrane and hydrophobic enough so that the hydrophobic membrane wouldn’t repel it. Membrane insoluble signals are hydrophilic and larger so a cell membrane receptor is needed in order for the cell to be able to detect them since they would be unable to pass through the membrane itself to relay that signal. Here is a good resource for this question, and also check out the pre-recorded lectures and topic notes: https://www.khanacademy.org/science/biology/cell-signaling/mechanisms-of-cell- signaling/a/signal-perception Question 3: Signal Amplification, Speed of Signaling and Crosstalk A. A single signal binds to an external receptor on the cell, but many downstream effector molecules inside the cell may end up, and need to be, activated in response to this single signal. This concept is called “signal amplification”. This process can be important for increasing the speed of transduction from a single signal, because it increases the concentration of active downstream molecules in the pathway, which means they will be more likely to find and bind to the next effector molecule in the pathway. **Remember that molecules “talk” to one another in the cell by physically binding to each other and that their movement inside the cell is (usually) by random diffusion. This means that concentration of these molecules can really affect how quickly they manage to bump into each other and cause the next step to happen in the pathway. Now describe the concept of signal amplification, then draw some sort of drawing or diagram to go with your description.

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Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis Signal amplification is when a multitude of a molecules are produced inside of the cell in response to a signal activating the receptor. The activated receptor “amplifies” the signal it has received by activating enzymes that can quickly produce more of the molecules corresponding to the signal. This increases the concentration of these molecules, and this could be necessary to increase the speed that the next reaction that requires this molecule can occur. The speed of the next reaction is increased because now that there is a higher concentration of this molecule there is a higher chance that it will interact and bind with the proteins it is intended to bind to.

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis B. Signal transduction pathways are typically a linear sequence of events – sort of like putting down a row of dominos, tipping the first one into the second one, which causes the second one to tip into the 3 rd one, etc. Sort of like this: https://www.youtube.com/watch?v=NlHAW1YTWiM (this is an excessive number of dominos though ☺ ) Sometimes signal transduction pathways want to integrate or coordinate their signaling and so they use crosstalk to do that. What is meant by “crosstalk” in this context? https://en.wikipedia.org/wiki/Crosstalk_(biology) Crosstalk refers to when results of one signal transduction pathway interfere or interact with other signal transduction pathways that are occurring within the cell. Some of these components that interact with other pathways can help the cell coordinate or change parts of one of the pathways so that they can both work together. C. Across organisms, and even within a single cell, there are many, many different types of signal transduction pathways. Some pathways have many components or steps in the pathway before the pathway outcome occurs, and some pathways don’t have many steps. Each pathway has evolved to function in the way that makes the most sense for that pathway and for the cells. To further delve into the concepts of cross-talk, signal amplification, and speed of signal transduction, I’d like you to discuss whether a longer or more complex pathway would work better for each of those mechanisms, or if an abbreviated, shorter pathway would work better. A. Do you think there would be more opportunity for crosstalk in a two-component pathway or a seven-component pathway? Why? There is more opportunity for cross talk in a seven-component pathway because there are more signal transduction pathways occurring. If there are multiple pathways occurring, each activating their own sets of enzymes and proteins it is more likely that those will somehow interact with the components of different pathways simply due to the sum of components that would be set off and the number of pathways that are in process.

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis B. Do you think there would be more opportunity for signal amplification in a two- component pathway or a seven-component pathway? Why? Seven-component pathway because there are more messengers that need to be produced downstream than a two-component pathway would need. If there is seven components to the pathway it is more likely that one of these components would cause an amplification of molecules to reach other components that need to be activated. C. Do you think the signal be transduced faster in a two-component pathway or a seven-component pathway? Why? Two-component pathway because there are less components that need to be activated in order to provide the outcome that the signal intended to provide. Question 4: Kinases and Phosphorylation Kinases are very common signal transducing/regulatory enzymes in the cell. In reference to when a kinase phosphorylates a protein in a eukaryotic cell, answer the following questions: A. What does a phosphate group look like? Describe it and draw it (yourself). A phosphate group is a phosphorous atom bonded to 4 oxygen atoms. It is very polar and negatively charged as one of these bonds is double stranded and the others 3 are negatively charged.

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Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis B. To what does the phosphate become attached when a molecule (usually proteins but not always) is phosphorylated? Which amino acids in proteins in eukaryotes are typically phosphorylated and what about their structure allows them to be phosphorylated? The phosphate attaches to the OH (hydroxyl) group on molecules. Common proteins that are phosphorylated are serine, threonine, and tyrosine. This is because these proteins have an OH group that is positively charged and the phosphate group is negatively charged. An H molecule is usually the byproduct because O can only make two bonds. C. Is this reaction reversible (ie can the phosphate be taken off after it gets put on) and if so, is there an enzyme that performs this action? Explain. Yes, there is an enzyme called phosphatase whose job is to remove phosphate groups from the proteins who bonded with them in order to inactivate them. The phosphate group that attaches to the proteins change their shape which in turn changes their function and phosphatase is crucial in being able to regulate protein function. D. What type of bond occurs between the phosphate and the protein (covalent or non- covalent and why does this matter)? Explain. The bond between phosphate and the protein is a covalent bond because atoms are being shared between the two. These bonds are strong and solidify that the phosphate group stays attached to the protein in order for it to remain activated as intended. E. What is the source of the phosphate in eukaryotes? Draw (by hand) the structure of that molecule. ATP is the source of phosphates in eukaryotes.

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis F. What are some typical changes that occur to protein activity due to phosphorylation? How does phosphorylation lead to these changes in protein activity? Explain. Phosphorylation causes the chemistry of the molecule to change. They could change the shape of the molecule/protein which leads to a change in function of that molecule/protein. The actual change in activity in the molecule depends on what molecule it is, it can lead to activation, deactivation, or change the proteins binding characteristics. Question 5: General Principle of Cell Signaling and Signal Transduction Look over the following statements about signal transduction and determine whether they are true or false. If you think they are false then re-write the sentence to make it a true statement or explain your reasoning. A. There is never a time in which more than one signaling pathway is functioning at the same time. False. There are times when there are many signaling pathways functioning at the same time. This is because the cell have many different receptors and can be receiving many different signals from the outside of the cell at the same time. B. Cells in the human body that aren’t r eceiving any signals remain quiescent (meaning inactive but alive) False. A cell in the human body will undergo cell programmed death when they are not receiving signals This is because cells need to receive survival signals in order to remain alive, otherwise the cell is programmed to undergo cell death. C. The same signal can lead to different outcomes in different cell types in the body True. This is because different cell types contain different proteins and molecules and receptors within them that changes what the signal does to that cell. D. Receptor-mediated endocytosis always results in degradation of cell surface receptors. False. Receptor-mediated endocytosis does not always result in degradation of cell surface receptors. Sometimes the signal attached to the receptor is stripped off and then the receptor is brought back to the cell surface for it to be re-used.

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis E. Phosphorylation of a protein changes its amino acid sequence. False. Phosphorylation does not change the proteins amino acid sequence. The protein remains intact, but its function could change because the addition of a phosphate changes its overall shape which changes how that protein interacts with its surroundings and other proteins meaning that its function can be changed. The phosphate is not an addition of amino acids to the protein. A Simplified Version of an Example Signal Transduction Pathway Diagram 1: Question 6: Self-Renewal Signaling Diagram The above simplified example of an actual signaling pathway is based off of the RTK- Ras-MapK pathway, which is often used in cell division signaling (aka self-renewal signaling). Use the basic signaling diagram (Diagram 1) to answer the following questions: A. (i) Do the components of the signaling pathway have to already be expressed (transcribed and translated) and present in the cell for the cell to receive and transduce the signal? (ii) Is there typically more than one copy of each of these pathway components expressed

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Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis (i) Yes the components need to be present in order for the signal to be transduced because each component leads to the activation or phosphorylation of another component which eventually leads to the outcome that the signal is meant to produce. Without one of these components then the signal would not be expressed since the pathway for it to be expressed is not complete. (ii) Yes, there is many copies of each component in the pathway. This is because the components are usually free floating in the cell so they will need many of them for the likelihood of the next component to be activated to increase. B. Describe each step of this signal transduction pathway in Diagram I. Remember that these pathways are like a set of falling dominos – the previous domino has to fall for the next domino to fall, which means that each step is dependent on the previous step occurring. Describe what needs to happen for the next step to happen (feel free to number of bullet point your response): - The first step in signal transduction is receiving the signal. When the signal is received, two receptors come together in a process called dimerization. This allows the receptors to be activated as kinases (allowing them to bind to ATP) on the inside of the cell and they can phosphorylate each other. - The phosphorylation of the receptors then causes TK1 (tyrosine kinase 1)to become phosphorylated and active. - TK1 is a kinase that can bind ATP and TK2. This means that once TK1 is active, it can bring ATP to TK2 and phosphorylate it. - When TK2 is phosphorylated it changes in a way that develops an affinity for TFa (transcription factor a) and ATP. So it can bring ATP to the TFa and activate TFa. - TFa is a transcription factor which means it regulates transcription. So once it is phosphorylated and activated by TK2 it enters the nucleus and regulates gene expression by activating transcription of that gene. - Gene expression is the outcome of the signal in this pathway. Question 7: The Mechanism of RTK Activation This series of questions refers to the mechanism of RTK activation. Here is a good resource for this: https://www.nature.com/scitable/topicpage/rtk-14050230/

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis A. If a mutation occurs such that the signal molecule cannot dimerize, will RTK be phosphorylated? No because dimerization is the process that brings two RTKs together so they can serve as a functional unit. When they are separate, they are not functional because they can only phosphorylate and activate each other. One RTK cannot phosphorylate itself. B. If a mutation occurs such that several of the tyrosine’s on RTK cannot be phosphorylated, will RTK dimerize? Yes, it will dimerize because it is the signal that causes RTKs to associate with each other and come together (dimerization). However, it will not be active as a kinase and it will not phosphorylate itself or any other components that were a part of the signal transduction pathway, so the signal will never be expressed in the cell. Questions 8: Broken Self-Renewal Signaling Pathway You have a stem cell that fails to respond when presented with a self-renewal signal. The stem cell appears to be permanently insensitive to the signal molecule (meaning no signal transduction outcome in response to the signal). Which of the following could explain the behavior of this cell? Yes or no and explain your reasoning for each question. A. There is a mutation in the kinase domain of the receptor Yes, this would prevent the signal from being expressed because the kinase domain in the receptor is what phosphorylates the RTK’s. If the receptor is not phosphorylated it will not be able to phosphorylate TK1 which t hen won’t activate to be able to phosphorylate TK2 which won ’t be able to activate TFa which then won’t be able to travel to the nucleus in order to activate gene expression. B. There is a mutation in TK2 such that it cannot be phosphorylated by TK1 Yes, this would prevent the signal from being expressed because TK2 must be able to bind to TK1 in order for TK1 to be able to bring ATP to TK2 so that it can be phosphorylated and activated. C. There is a mutation in TK2 such that it is always active as a kinase No, this would not prevent the signal to be expressed because the signal leads to the activation of TK2 anyways by signal transduction. If it is already activated it should not

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis be a problem as long as TK1 is still able to bring ATP to it so it can transfer ATP to the TFa. D. The cell surface receptor that binds this signal is not being expressed and so is not present on the surface of the cell. Yes, this would prevent the signal from being expressed because The signal would never be received by the cell. If the receptor is never activated by the signal than transduction of that signal will never begin. Question 9: Signal Transduction and Feedback Loops Feedback loops are used very commonly both in cell physiology as well as whole organism physiology (in multi-cellular organisms like us). A. What is a feedback loop (give an explanatory definition) as it pertains to a signal transduction pathway? How is a positive feedback loop different from a negative feedback loop? These are definitions so describe them as such. A feedback loop is when a downstream component from a linear pathway regulates an earlier component in that same pathway. A positive feedback loop regulates the signal transduction by increasing the amount of activity of the earlier component, while a negative feedback loop decreases the activity of the earlier component lessening the signal transduction. B. Using our hypothetical signal transduction pathway from class, draw and describe an example of a positive feedb ack loop and a negative feedback loop. You’ll probably need to add in additional proteins and cellular processes to create your loops so feel free to use your imagination and what you’re learning in this unit to do that.

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Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis C. More on feedback loops …. (i) If the outcome (in this case, activation of gene expression) of a signal transduction pathway led to activation or production of a protein involved in ubiquitin-mediated proteolysis targeting say, TK1, what would be the result and do you think this would be an example of positive feedback, negative feedback or not a feedback loop? Explain your reasoning. A positive feedback loop would result in higher activity of production of the protein that targets TK1 into ubiquitin-mediated proteolysis, which would in turn degrade many TK1’s resulting in lower expression of another signal that induces gene expression that begins self-renewal. A negative feedback loop would lead to less production of these proteins that target TK1’s meaning that there will be many TK1’s to transduce the signal that leads to gene expression of self-renewal in the cell. No feedback loop means that the signal to create these TK1 targeting proteins will continue until the signal is not present. (ii) What about receptor-mediated endocytosis? (As in: outcome of pathway leads to activation or production of protein that leads to endocytosis of the pathway’s receptor). As always, fully explain your reasoning. A positive feedback loop will result in an increased production of these proteins that induce receptor-mediated endocytosis which will lead to less of the signal being received since there will be less receptors to receive the signal. This means less of these proteins will be made once the cell is unable to receive the signal for this protein productions. A negative feedback loop will decrease the production of proteins that induce receptor endocytosis, so the cell will slowly stop receiving this signal because these proteins will still be produced just at a slower rate until no more receptors are available to receive the signal that induces the production of these proteins. No feedback loop would mean that the cell will receive the signal and produce these receptor endocytosis proteins at a normal rate until no receptors are left on the cell surface.

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis (iii) If the outcome (in this case, activation of gene expression) of a signal transduction pathway led to activation or production of a protein involved in phosphorylation and activation of TK2, what would be the result and do you think this would be an example of positive feedback, negative feedback or not a feedback loop? Explain your reasoning. A positive feedback loop would result in the amplification of TK2, because there will be more activated TK2 around. This would cause an increase in speed of gene expression that caused by TFa because TK2 phosphorylates TFa and activates so it can express the self-renewal gene. A negative feedback loop would result in less of the proteins that activate TK2 being made, but it is still slowly increasing the speed in which TK2 is being phosphorylated and expressed because these proteins are still being made just at a slower rate. No feedback loop would result in a nonstop expression of TK2 until the signal is gone because this protein the rate of this proteins production would not stop. Question 10: Problem Solving in a Signal Transduction Pathway You perform an analysis of the pathway in Diagram 1 with the assumption that there are other components and pathways around in the cell. You see the following results: Present? Phosphorylation state Signal Yes (in environment surrounding cell) Not applicable Receptor No Not applicable (because receptor isn’t present) TK1 Yes Yes TK2 Yes No TFa Yes Yes Response occurring Yes Not applicable The table above shows the status of several important factors in signal transduction in a given moment of time in a stem cell. Remember that this is a snapshot of a moment in time and that signal may have been bound previous to this moment in time and may have activated the pathway already. Treat each question independently of the others and please be sure to thoroughly explain your reasoning.

Bio151 Sp23 Topic 6 Cell Signaling and Signal Transduction Problem Set Francis A. What happened to the receptor? By what mechanism can receptors be removed from the surface of cells? Explain. The receptor was brought into the cell by a mechanism called receptor mediated endocytosis. When the signal is detected some receptors are brought into the cell because the signal would have somehow changed the chemistry of the receptor. However the signal is not present, this could be because the signal was already removed from the cell in order for the receptor to be re used or degraded. B. Why do you still see outcome occurring despite the fact that some components of the pathway are missing or not active? Explain. There might be a mutation in the cell in which TK1 is allowed to bind with TFa in order to bind ATP to it and phosphorylate it. There are also many other ways this could’ve happened as the cell has multiple pathways and mechanisms it can use that cross talk with each other in order to make sure everything is working smoothly. C. Is it possible that ubiquitin-mediated proteolysis has removed the receptor from the cell surface? Explain. It is possible that ubiquitin-mediated proteolysis has removed the receptor because the receptor has a intra cellular section inside of the cell and ubiquitin is transferred through protein interaction, so if a protein that carries ubiquitin “tagged” the intracellular section then the whole receptor would be dragged by the ubiquitin to the proteolysis to be degraded. D. Assuming that TK2 was phosphorylated at some moment in time, by what enzymatic activity has it been dephosphorylated? Phosphatase is the enzyme that dephosphorylates proteins and molecules. E. If TK2 is dephosphorylated, hypothesize how TFa could still be phosphorylated and that response to the signal is still occurring. There are several possibilities. Feel free to include methods of modulation that we’ve discussed, such as feedbac k loops, proteolysis, endocytosis or pathway crosstalk. Explain your reasoning. There could have possibly been a positive feedback loop that would allow TFa to stay phosphorylated if a downstream component was introduced that would phosphorylate TFa. Then TFa would not need TK2 to be activated in order for the gene to be expressed.

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AVPL Problem Set 6 COMPLETE

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