Differential Signaling of Cyclic AMP
Furthermore, down-regulation of RIIβ leads to increased AKT/PKB .. we also find a close connection between PKA cAMP sensitivity and levels. University of Pennsylvania Medical Center, Curie Blvd., BRB II/III, However, no significant difference was observed when intact sperm were examined. When the sperm-specific catalytic subunit of PKA is eliminated by Phospho-Akt [Ser ]; Cell Signaling, Danvers, MA; for blot). PKB activation and enhanced GC resistance in C7–14 cells. These results There are two major isoforms of PKA, designated as PKA(I) and. PKA(II) Toxicology, The University of Texas Medical Branch, University Blvd.,. Galveston . cAMP and GC in CEM Cells—Although the connection between.
Therefore, it is imperative to reformulate concepts of cAMP-mediated signaling to include the contribution of Epac. The existence of Epac immediately raises many questions regarding the mechanism of cAMP-mediated signaling.
Since both PKA and Epac are broadly expressed in many tissues 1819an increase in intracellular cAMP levels will lead to the activation of both enzymes, and possibly other potential cAMP target s as well.
Differential Signaling of Cyclic AMP
It is conceivable that while PKA acts through a discrete set of signaling pathways, Epac may enlist distinct signaling pathways, and the net cellular effects of cAMP are dictated by the sum of these events. Therefore, the apparent cellular effect of cAMP can vary depending upon the relative cellular abundance and distribution of Epac and PKA.
Our hypothesis is that there is a dynamic control of cellular expression and targeting of Epac and PKA which, coupled with the dynamic changes in the concentration of cAMP, are the controlling mechanisms that determine the observed cell type-specific cAMP effects. Our study shows for the first time that although Epac and PKA are activated by a common upstream second messenger cAMP, they can exert opposing effects in regulating downstream targets such as PKB.
Monoclonal Rap1 antibodies were purchased from Transduction Laboratories. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgGs were from Bio-Rad. Culture media were from Invitrogen.
Fetal bovine serum was from Sigma. All chemicals were reagent grade. Antibody elution fractions were dialyzed in PBS. Specificity of purified antibody was confirmed by Western blot using recombinant Epac protein expressed in insect Sf9 cells. Stable transfectants were selected using G at 0. Activation was terminated by washing cells twice with PBS.
Cell lysates were prepared by directly suspending the cells into Laemmli SDS sample buffer Membranes were then washed with TTBS and incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibody for 1 h at room temperature. Following three min washes in TTBS, membranes were treated with substrates for 5 min and exposed to x-ray film at room temperature. So it can be in equilibrium with-- since it's a weak acid, it's going to produce some hydrogen proton.
And then the rest of the molecule is going to keep the electrons. So it's going to be plus-- oh, this is in an aqueous solution. Let me do that. And then you're going to have the rest of the acid, whatever it might be. A minus, and that's also going to be in an aqueous solution.
pKa and pKb relationship (video) | Khan Academy
And that's the general pattern. We've seen the case where A could be an NH3, right? And this is just NH3. A could be a fluorine molecule right there, because then this would be hydrogen fluoride or hydrofluoric acid. And this would just be the negative ion of fluorine. Or a fluorine with an extra electron. So it could be a bunch of stuff. You can just throw in anything there, and it'll work.
Especially for the weak acids. So we learned a last video that if this is the acid, then this is the conjugate base. And we could write the same reactions, essentially, as kind of more of a basic reaction. So we could say, if I start with A minus-- it's in an aqueous solution-- that's in equilibrium with-- This thing could grab a hydrogen from the surrounding water and become neutral then. It's still in an aqueous solution.
Relationship between Ka and Kb
And then one of those water molecules that it plucked that hydrogen off of is now going to be a hydroxide molecule. Remember, whenever I say pluck the hydrogen, just the proton, not the electron for the hydrogen. So the electron stays on that water molecule, so it has a negative charge. It's in an aqueous solution.
So we could write the same reaction both ways. And we can write equilibrium constants for both of these reactions. So let's do that. Let me erase this, just because I can erase this stuff right there, and then use that space. So an equilibrium reaction for this first one. I could call this the K sub a, because the equilibrium reaction for an acid. And so this is going to be equal to its products. So the concentration of my hydrogen times my concentration of whatever my conjugate base was, divided by my concentration of my original acid.
So this would be the concentration of HA. I could also write an equilibrium constant for this basic reaction. Let me do it right down here. So I'll call that my K sub b. This is a base equilibrium. And so this is equal to the concentration of the products. It's becoming tedious to keep switching colors.
Actually, I'll do it.
Because it makes it easier look at, at least for me. HA times the concentration of my hydroxide ions divided by my concentration of my weak base. Remember, this can only be true of a weak base or a weak acid. If we were dealing with a strong acid or is or a strong base, this would not be an equilibrium reaction. It would only go in one direction. And when it only goes in one direction, writing this type of equilibrium reaction makes no sense-- or equilibrium constant-- because it's not in equilibrium.
It only goes in one direction. If A was chlorine, if this was hydrochloric acid, you couldn't do this.
Relationship between Ka and Kb (article) | Khan Academy
You would just say look, if you have a mole of this, you're just dumping a mole of hydrogen protons in that solution and then a bunch of chlorine anions who are not going to do anything. Even though they are the conjugate base, they wouldn't do anything. So you can only do this, remember, for weak acids and bases.
- pKa and pKb relationship
So with that said, let's see if we can find a relationship between Ka and Kb. What do we have here? We have an A minus on both sides of this. We have H over-- OH over A minus. Let's solve for A minus. If we multiply both sides of this equation by HA over H plus, on the left-hand side we get Ka times the inverse of this. So you have your HA over H plus is equal to your concentration of your conjugate base.
And let's do the same thing here. Solve for A minus. So to solve for A minus here, we might have to do 2 steps. So if we take the inverse of both sides, you get 1 over Kb is equal to A minus over H, the concentration of my conjugate acid times the concentration of hydroxide.
Multiply both sides by this. And I get A minus is equal to my concentration of my conjugate acid times concentration of hydroxide. All of that over my base equilibrium constant. Now, these are the same reactions. In either reaction for given concentrations, I'm going to end up with the same concentration. This is going to equal that. These are two different ways of writing the exact same reaction. So let's set them equal to each other.
So let me copy and paste it, actually. So I'm saying that this thing, copy, is equal to this thing right here. So this is equal to-- let me copy and paste this-- that. That's equal to that. So let's see if we can find a relationship between Ka and Kb. Well, one thing we can do is we can divide both sides by HA.
So if we divide both sides by HA. Actually, I could probably have that earlier on to the whole thing. If we ignore this part right here, this is equal to that. Let me erase all of this. I'm using the wrong tool. So we could say that they both equal the concentration of A minus. So that's equal to that. We can divide both sides by HA. This over here will cancel with this over here.
And we're getting pretty close to a neat relationship.