cAMP & PKA: Videos & Practice Problems

In the GPCR signaling pathway, the enzyme adenylate cyclase converts ATP into the secondary messenger cAMP, which activates protein kinase A (PKA). PKA, a serine-threonine kinase, phosphorylates target proteins, influencing cellular responses. cAMP is inactivated by cAMP phosphodiesterase, converting it to AMP, which does not activate PKA. Additionally, serine-threonine phosphatases reverse PKA activity by removing phosphate groups, resetting the signaling pathway. This intricate regulation ensures precise control over cellular functions and responses.

cAMP & PKA

So here we're going to briefly revisit our map of the lesson on biosignaling pathways which is down below right here. And, of course, we've been exploring this map by exploring the leftmost branches first, and so we've already introduced G protein-coupled receptors or GPCRs, and we've talked specifically about the stimulatory adenylate cyclase GPCR signal transduction pathway. And so here in this video, we're going to continue to talk more and more about this pathway by talking about the secondary messenger cAMP, and the enzyme protein kinase A or PKA. And so let's go on and get started talking about these.

So now that we've covered stimulatory adenylate cyclase GPCR signaling, here we're going to continue to talk about that same pathway by focusing on the secondary messenger cAMP and the enzyme protein kinase A or PKA for short. And so in this video specifically, we're going to focus on the production and the function of the secondary messenger cAMP. And so recall from our previous lesson videos that the effector enzyme Adenylate Cyclase actually converts its substrate ATP into the secondary messenger cAMP, which is really just an abbreviation for cyclic adenosine monophosphate. So you can see cAMP, comes from that name.

And so if we take a look at our image down below over here on the left-hand side, notice on the left, we're showing you the structure of ATP, which we know again is the substrate for the effector enzyme Adenylate Cyclase. And so notice here as the catalyst to this particular reaction that we're showing, we have the enzyme Adenylate Cyclase in its active form. And so when Adenylate Cyclase is activated, it can actually convert its substrate ATP into the secondary messenger that we see over here, cAMP. And so you can compare the structure of cAMP with ATP to get a good idea that they are related to one another. So notice that P2 inorganic phosphates here are released and, in the process, the phosphate here is cyclic, which is why it's called cyclic adenosine monophosphate because it only has P1 phosphate group that is in a cyclic form as we see down below.

Now really this secondary messenger molecule that we see here, cAMP, again is a secondary messenger that goes on to activate the enzyme that's called cAMP-dependent protein kinase A or just PKA for short. And so you can see PKA comes from this abbreviation for protein kinase A, and this is a cAMP dependent enzyme. And so PKA is only going to become active, in the presence of the cAMP secondary messenger. And so, therefore, what we can say here is that cAMP is going to function as an allosteric activator, which recall that we can symbolize using a positive sign for activation. And so cAMP is an allosteric activator for the enzyme protein kinase A.

And so if we take a look at our right side of the image over here, notice that we're showing you the activation of the enzyme PKA. And notice that we're showing you specifically P4 cAMP molecules. And these P4 cAMP molecules will bind to the inactive form of PKA. And so, when cAMP binds to the inactive form of PKA, as we see above, the cAMP molecules are these little guys that we see when they bind to the inactive form of the PKA, it actually activates the subunits of PKA. And we're going to talk in more detail specifically about this process right here and the activation of PKA in our next lesson video. But for now, what you can see is that cAMP is acting as an allosteric activator for PKA to activate it. And so, this here concludes our introduction to the production and function of cAMP, and I'll see you guys in our next video.

cAMP & PKA

In this video, we're going to talk about the activation of cAMP-dependent protein kinase A or PKA for short. Now recall from our previous lesson videos that kinases are enzymes that utilize energy in the form of ATP in order to phosphorylate their substrates or add a phosphate group to their substrates. And of course, protein kinase A is indeed a kinase itself, so it is going to phosphorylate its substrates using ATP. Now what's also important to note is that in the absence of any secondary messenger cAMP, the enzyme Protein Kinase A or PKA is in its inactive heterotetramer form. And so hetero we know is a prefix that means different, tetra is a prefix that means 4, and so that means it's going to have 4 subunits that are different from each other. They're not all identical. And it turns out that it actually has 2 regulatory subunits and 2 catalytic subunits. So it's referred to as R₂C₂ where the R represents the regulatory subunits and there are 2 of them, and the C represents the catalytic subunits and again there are 2 of them. And so if we take a look at our image down below, notice up here at the top left we have a little reminder of our adenylate cyclase GPCR signaling pathway. So we have our hormone signaling ligand epinephrine or adrenaline will bind to the GPCR, more specifically the beta adrenergic GPCR, causing a conformational shift in this GPCR that ultimately activates the G protein that we have down below, where it's going to promote it to exchange its low energy GDP for a high energy GTP. And so that is going to promote the dissociation of the alpha subunit, which goes on to activate the effector enzyme adenylate cyclase, which we mentioned in our last lesson video converts the substrate ATP into the secondary messenger, cAMP. Now what you'll notice is, here, we're showing you the structure of the protein kinase A before cAMP has an effect on its structure. And so we said that PKA exists as an inactive heterotetramer with 4 subunits that are not identical, more specifically 2 regulatory subunits which we abbreviate with R's, and 2 catalytic subunits which we abbreviate with C's. And so when PKA is in this heterotetramer form, it's in its inactive form, so it will not be performing its function. Now what's important to note is that cAMP is going to act as an allosteric activator to Protein Kinase A, and more specifically 4 cAMP molecules are needed to bind to the regulatory PKA subunits, and that is going to release the 2, the 2 catalytically active PKA subunits. And so these catalytically active PKA subunits, of course, are going to be able to phosphorylate their substrates. More specifically, PKA is a serine-threonine kinase, so, it will be able to phosphorylate serine-threonine residues on their target proteins in order to alter the activity of their target proteins, and ultimately the altered activity of the target proteins leads to the cell response. And so if we take a look at our image down below, notice that it specifically takes 4 cyclic AMP molecules in order to interact with the inactive PKA to generate the active form of PKA over here. And so notice that we have these 4 cAMP molecules here that bind to the regulatory subunits of PKA, and that causes the dissociation of the regulatory subunits and releases the 2 catalytically active subunits that we can abbreviate with C's down here. And these 2 catalytically active PKA subunits can then act as Serine-Threonine Kinases to phosphorylate their substrates. And so what that means is they could take an inactive enzyme and phosphorylate it to create an active enzyme that would lead to the cell response, or again since phosphorylation does not necessarily always mean activation, it could take an active enzyme that is not phosphorylated and phosphorylate it to create an inactive enzyme, and that would also lead to the cell response. But ultimately, the main takeaway here is that it takes 4 cAMP molecules to activate the 2 catalytic PKA subunits. And so really this here is the conclusion of how cAMP-dependent protein kinase A or PKA is activated, and we'll be able to get some practice applying these concepts as we move forward in our course. So I'll see you guys in our next video.

cAMP & PKA Example 1

Alright. So here we have an example problem that wants us to order each of the steps in the activation of PKA by numbering them 1 through 4, and notice the first step is already numbered for you, starting immediately after adenylate cyclase activation. And so notice that immediately after adenylate cyclase activation, we're told that the very first step here is that cytosolic cAMP concentration increases, and so what we need to do is number each of these other three steps that we see down below in the proper order using numbers 2, 3, and 4. And so of course immediately after adenylate cyclase activation, after cytosolic cAMP concentration increases, the next thing that's going to happen is that cAMP is going to be able to act as an allosteric activator to PKA. And so 2 cAMP molecules will bind to each of the PKA regulatory subunits, where we know that there are 2 regulatory subunits. So really there are 4 total cAMP molecules needed to regulate the PKA subunits. And so this event here will be the second event to take place. And then after these cAMP molecules bind to the regulatory subunits, the next event is that the regulatory subunits of PKA need to dissociate from the catalytic subunits so that the catalytic subunits can be released in their active forms. And so what we're seeing here is that this is going to be the third event that occurs immediately after this. And then of course, last but not least, what we have is the last step, step 4 is right here, which is that the free catalytic subunits that were released can then phosphorylate proteins on their serine and threonine residues. And so we can go ahead and label this as step 4. And so really this is the answer to this practice problem and I'll see you guys in our next video.

Which of the following statements about protein kinase A (PKA) is false?

• A. PKA binds a total of four molecules of cAMP, two molecules on each of the regulatory (R) subunits.
• B. PKA binds a total of four molecules of cAMP, one on each of the four subunits.
• C. Once active, the catalytic (C) subunits dissociate and phosphorylate target proteins.
• D. When inactive, PKA is a tetramer of two regulatory (R) and two catalytic (C) subunits.

Answer: (B)

cAMP & PKA

So now that we've talked about the production of cAMP and the activation of PKA, in this video, we're going to introduce the inactivation of the secondary messenger molecule cAMP, and the inactivation of the enzyme PKA. And so recall from our previous lesson videos that the GTPase activity of the G Protein Alpha subunit is ultimately what is going to inactivate or terminate the GPCR signaling pathway to help reset that entire pathway. But what we did not get to mention in our previous lesson videos is that there are also 2 other events that also allow for the termination of the GPCR signaling pathway, and those two events are going to be the inactivation of cAMP and the inactivation of PKA. And so we've separated out these 2 events into event number 1 and event number 2 down below, and notice that these events also correspond with the events that you see down below labeled 12. And so again, the first event here is going to be the inactivation of cAMP. And so cAMP signaling effect can actually be turned off by decreasing the concentration of cAMP within the cell, and the cell can decrease its concentration of cAMP using the enzyme known as cAMP phosphodiesterase. And so cAMP phosphodiesterase is an enzyme that is going to convert the secondary messenger molecule cAMP into its non cyclic version AMP. And so notice that the really difference between, the real difference between cAMP and AMP is the c here that is not present in AMP. And so recall that the c represents cyclic and really that's all that's changing with AMP and we'll be able to see that down below in our image. But the biggest thing to note is that this AMP actually does not activate PKA like what cAMP does, and so it's actually a big deal to convert cAMP into AMP. And, of course, this, by converting cAMP into AMP, that's going to decrease cAMP concentration and help turn off the signal and, essentially help terminate the GPCR signaling pathway. And so if we take a look at our image down below, notice again up here at the top left, we have a little reminder of our stimulatory GPCR signaling pathway. So we know that the hormone, extracellular ligand epinephrine or adrenaline will bind to the beta-adrenergic GPCR causing a conformational shift that promotes GTP replacement of GDP, and that is going to allow the dissociation of the G alpha subunit to diffuse and activate the Adenylate Cyclase Effector Enzyme, and that will convert its substrate ATP into cAMP. And then, of course, cAMP here is going to promote, the cell response like we talked about in our previous lesson video. But if we want to turn off this effect, if we want to turn off the signaling pathway and reset the pathway, then we need to get rid of some of the cAMP and decrease the concentration within the cell to turn off the pathway. And so that's exactly where cAMP phosphodiesterase comes into play because it converts the cAMP molecule over here, the cyclic version of, the phosphate group here into AMP, and the most important part about AMP is that its phosphate group is not going to be in the cyclic version and so it's going to look like this. So we can go ahead and label that as the phosphate group. And so notice that it is not cyclic over here whereas over here it is cyclic, and so AMP is not able to regulate PKA like what cAMP is usually able to do. And so cAMP phosphodiesterase is able, again to help turn off the entire signaling pathway. Now moving on to step number 2, which is again going to be the inactivation of PKA, on the other hand, notice that PKA's activity is going to be reversed by Serine Threonine Phosphatases. And so phosphatases, you might recall from our previous lesson videos, are pretty much enzymes that are the opposite of kinases. And so Serine Threonine Phosphatases are going to remove phosphate groups from their substrates instead of add phosphate groups to their substrates like what kinases do. So if we take a look at our image down below, what you can see is that normally cAMP would bind to the regulatory subunits of PKA, which is again this is the inactive form of PKA over here, And, when the cAMP binds to the regulatory subunits, it would release the catalytically active, PKA subunits here, which, we can label these as active. And then these catalytically active PKA subunits, because they are kinases, they can phosphorylate enzyme substrates, and that will lead to the cell response. But, of course, even, with the activity of cAMP phosphodiesterase and even by cleaving the GTP in the, G protein, that is not going to remove the phosphate groups that are on these enzymes. And so if we really want to reset the pathway and turn off the signaling pathway, then we need a way to remove the phosphate groups, and that's exactly where the Serine Threonine Phosphatases come into play. And so over here in step number 2, what you can see is that the phosphatases will remove the phosphate groups that the PKA added to its targets. And so that is going to help revert back the entire process and help to reset the entire pathway, essentially helping to inactivate PKA further. And so really, this here concludes our introduction to the inactivation of cAMP and PKA, and we'll be able to get some practice with these concepts as we move forward in our course. So I'll see you guys in our next video.

What could be the result of a mutation in the R subunits of cAMP-dependent protein kinase A (PKA) that inhibits formation of the R₂C₂ protein complex?

• A. PKA will always remain in the inactive state.
• B. cAMP would drastically decrease PKA activity.
• C. PKA will always remain in the active state.
• D. No effective change occurs.

Answer: (C)

The image below is a schematic representation of PKA activation from epinephrine binding. Based on the provided numbers in the diagram, how many subunits of catalytically active PKA will there be?

• A. 1,000 molecules.
• B. 200 molecules.
• C. 100 molecules.
• D. 50 molecules.
• E. 400 molecules.

Answer: (C)

Here’s what students ask on this topic:

What is the role of cAMP in the GPCR signaling pathway?

In the GPCR signaling pathway, cAMP (cyclic adenosine monophosphate) acts as a secondary messenger. When a ligand binds to a GPCR, it activates the enzyme adenylate cyclase, which converts ATP into cAMP. The cAMP then activates protein kinase A (PKA) by binding to its regulatory subunits, causing the release of its catalytic subunits. These catalytic subunits phosphorylate target proteins, leading to various cellular responses. This process is crucial for amplifying the signal and ensuring a precise cellular response.

How does protein kinase A (PKA) become activated?

Protein kinase A (PKA) becomes activated through the binding of cAMP. In its inactive form, PKA is a heterotetramer composed of two regulatory and two catalytic subunits. When four cAMP molecules bind to the regulatory subunits, it causes a conformational change that releases the catalytic subunits. These free catalytic subunits are then able to phosphorylate target proteins, which can alter their activity and lead to various cellular responses. This activation mechanism ensures that PKA is only active when cAMP levels are elevated.

What enzyme is responsible for the inactivation of cAMP?

The enzyme responsible for the inactivation of cAMP is cAMP phosphodiesterase. This enzyme converts cAMP into AMP (adenosine monophosphate) by breaking the cyclic bond in cAMP. AMP does not activate PKA, thus reducing the cellular response initiated by cAMP. This conversion is crucial for terminating the signal and resetting the GPCR signaling pathway, ensuring that the cell can respond appropriately to new signals.

What is the function of serine-threonine phosphatases in the GPCR signaling pathway?

Serine-threonine phosphatases play a critical role in the GPCR signaling pathway by reversing the actions of protein kinase A (PKA). These phosphatases remove phosphate groups from the serine and threonine residues of target proteins that were phosphorylated by PKA. This dephosphorylation process helps to inactivate the target proteins and reset the signaling pathway, ensuring that the cellular response is appropriately terminated and the cell is ready for new signals.

How does adenylate cyclase contribute to the production of cAMP?

Adenylate cyclase is an enzyme that plays a pivotal role in the production of cAMP. When activated by the Gα subunit of a G protein, adenylate cyclase catalyzes the conversion of ATP (adenosine triphosphate) into cAMP (cyclic adenosine monophosphate). This reaction involves the removal of two phosphate groups from ATP and the formation of a cyclic bond in the remaining phosphate group, resulting in cAMP. This secondary messenger then goes on to activate protein kinase A (PKA), leading to various cellular responses.