cAMP and Protein Kinase A

Adrenaline never enters the liver cell it gives orders to. It binds a receptor on the outer face of the membrane and stays there, yet enzymes in the cytosol change what they are doing. Something made inside the cell has to carry the order. That something is cyclic AMP (cAMP), and the enzyme that reads it is protein kinase A (PKA). Once you know both, you can take on a question Prof. Deppmann puts to his graduate students: how can one cell with one messenger give two different responses?

Adrenaline stays outside

When Earl Sutherland began studying hormones, he later recalled, there was “a widespread feeling among biologists that hormone action could not be studied meaningfully in the absence of organized cell structure.” His laboratory at Western Reserve University in Cleveland tested that feeling on liver, where adrenaline switches on phosphorylase, the enzyme that starts glycogen breakdown.

The laboratory homogenized liver and spun it in a centrifuge. That gave two fractions: a pellet of particles that later work traced to fragments of the cell membrane, and a clear liquid called the supernatant, which held the phosphorylase. The paper, by Theodore Rall, Sutherland and Jacques Berthet, came out in 1957. Its logic fits in three lines.

• Hormone added to the supernatant alone: phosphorylase stayed off.
• Hormone added to the supernatant with a little of the pellet mixed back in: phosphorylase switched on.
• Hormone incubated with the pellet, then the liquid from that tube added to the supernatant: phosphorylase switched on.

And the liquid still worked after the whole tube was boiled.

Read each result as a clue. The supernatant answers the hormone only when some pellet is added back, so the hormone acts on the pellet, and the pellet hands something to the soluble enzymes. That something survives boiling and passes through a dialysis membrane, so it is small and it is not an enzyme. Nothing in either tube is alive, so whatever passed between them is chemistry.

The factor turned out to be an adenine nucleotide whose single phosphate is bonded to the ribose at two positions, the 3′ and 5′ carbons, which closes the molecule into a small ring. Its name is adenosine 3′,5′-monophosphate, or cyclic AMP. David Lipkin’s chemistry group at Washington University in St. Louis had isolated the same compound from ATP treated with barium hydroxide, with no hormone in the work, and later established its structure.

Sutherland called the hormone the first messenger and cAMP the second messenger. Let cAMP be your definition. A second messenger is a small molecule that the cell makes when the first messenger binds outside, and that moves through the interior where the hormone cannot go. The cell must also be able to destroy it quickly, or the signal would never end. And it amplifies: one bound hormone yields many cAMP molecules.

The idea: the cAMP level is a balance between a tap and a drain

The tap is adenylyl cyclase. The hormone-sensitive forms of this enzyme sit in the plasma membrane with the active site facing the cytosol. Adenylyl cyclase converts ATP to cAMP and releases pyrophosphate. The G protein Gs turns the cyclase up and Gi turns it down (how strongly depends on which form of the cyclase a cell makes), so two receptors can cast opposing votes on one enzyme. The G-protein cycle itself is in the lesson on GTPase switches.

The drain is phosphodiesterase, which cuts the 3′ bond of the ring and leaves ordinary 5′-AMP. 5′-AMP does not switch on PKA, so the message is erased.

While phosphodiesterase is working well below saturation, it removes cAMP at a rate proportional to how much is there. The level then settles where the two rates match:

steady-state [cAMP] = rate of synthesis ÷ rate constant of breakdown

Two things follow. First, you can raise cAMP by opening the tap or by narrowing the drain, and doing both multiplies. Theophylline, a drug that inhibits phosphodiesterase, strengthens the effect of hormones that work through cAMP, and Sutherland made that one of his tests for whether a hormone uses cAMP at all. Caffeine is a close chemical relative and inhibits phosphodiesterase in the test tube, although at the levels reached by drinking coffee its main action is thought to be blocking adenosine receptors. Second, a fast drain makes a brief signal. When the hormone leaves, cAMP falls on a timescale set by the drain. The lesson on turning signals off takes that further.

A kinase held shut by its own partner

The main reader of cAMP is PKA, which Walsh, Perkins and Krebs described in 1968 as a protein kinase that depends on cAMP. At rest PKA is a complex of four subunits. Two are catalytic subunits, each a complete kinase. Two are regulatory subunits, which keep the catalytic ones off.

How they do it is simple. PKA phosphorylates a serine or threonine that lies close after a pair of arginines in its target. Each regulatory subunit carries a short stretch that looks like such a target, and that stretch lies in the catalytic subunit’s active site, where a substrate would go. In type I regulatory subunits the serine position holds a residue that cannot accept a phosphate, so the stretch fits but is never phosphorylated. Biochemists call it a pseudosubstrate. Think of a blank key left in a lock: it fits and will not turn, so no working key can get in.

Each regulatory subunit also has two binding sites for cAMP, which makes four in the whole complex. When cAMP fills them, the regulatory subunit changes shape and lets go. In the classical model the catalytic subunits come away free and begin phosphorylating targets. There is evidence that PKA held on an anchoring protein can act on nearby targets without fully coming apart, so take “the catalytic subunits float away” as the textbook picture, which may not hold everywhere.

We can describe the blocked active site because of Susan Taylor. In 1991 her laboratory and Janusz Sowadski’s crystallography group published the first three-dimensional structure of any protein kinase, the PKA catalytic subunit. The fold has two lobes. ATP binds under the small lobe, the substrate lies along the large lobe, and the phosphate is transferred in the cleft between them. In that crystal the substrate groove was filled by a 20-residue piece of a separate inhibitor protein, itself a pseudosubstrate. Later structures from Taylor’s laboratory showed a regulatory subunit’s own inhibitor stretch in the same groove. The human genome encodes about 500 protein kinases, and their catalytic cores are variations on this two-lobed fold.

What PKA phosphorylates depends on the cell

A free catalytic subunit phosphorylates the suitable targets it meets, and cells differ in which targets they make.

• Liver. PKA phosphorylates phosphorylase kinase, which switches on glycogen phosphorylase; the overview lesson on signal transduction walks through that cascade. PKA also phosphorylates glycogen synthase, which shuts glycogen synthesis down.
• Heart. PKA phosphorylates phospholamban, releasing a brake on the pump that returns calcium to its internal store, and troponin I on the contractile filaments. PKA activity also increases calcium entry through L-type calcium channels. The result is a stronger beat and a faster relaxation.
• Fat. PKA phosphorylates hormone-sensitive lipase and perilipin, a protein that coats the fat droplet, and stored fat is broken down.

Over a longer time, catalytic subunits that reach the nucleus phosphorylate the transcription factor CREB, which changes gene expression.

PKA is also one of several proteins that read cAMP. Epac is an exchange factor for the small GTPase Rap that cAMP activates directly, and cyclic-nucleotide-gated ion channels, such as those in olfactory neurons, open when cAMP binds them.

One messenger, many meanings

Half of the answer to the opening question is now plain. The effect depends on the proteins inside each cell. The messenger and the kinase are the same, and the substrates differ.

The other half is harder: two cAMP signals inside one cell. Heart muscle supplied the classic case. Hayes, Brunton and colleagues reported that kinase activity in the heart depended on which hormone was used. Isoproterenol, which acts on β-adrenergic receptors, and prostaglandin E1 both raised cAMP, but only isoproterenol activated the particulate pool of PKA, the part that pellets with the cell’s structures. In 1983 Buxton and Brunton named the result: compartments of cAMP and protein kinase. About twenty years later Zaccolo and Pozzan imaged cAMP in stimulated heart cells from newborn rats and saw small regions of high cAMP.

So cAMP does not fill a cell the way water fills a glass. Up to three things can work together to make a compartment:

1. The messenger moves slowly through the crowded interior of the cell.
2. Proteins that bind the messenger, PKA among them, may hold it near where it was made.
3. Phosphodiesterases sit where the cell needs the concentration to fall.

Anchoring proteins (the AKAPs from the lesson on adaptors and scaffolds) supply the addresses. They hold PKA beside a particular substrate, often with a phosphodiesterase in the same complex.

How much each mechanism matters is still disputed. Agarwal, Clancy and Harvey found that buffering, meaning that PKA binds cAMP and holds it, helps explain the slow movement of cAMP in heart cells. Richards and colleagues, working in adult ventricular myocytes, also found slow movement but explained it by tortuosity, the winding paths through a crowded cell, because cAMP moved no faster when they saturated the binding sites. Several studies also argue against a common textbook idea, that phosphodiesterases alone explain cAMP gradients. The compartments are observed. Their mechanism is not settled.

Design problem

A heart cell and a liver cell both raise cAMP when adrenaline arrives. Using the same messenger and the same kinase, design how the heart cell contracts harder while the liver cell releases glucose. Then say how one cell could keep two cAMP signals from mixing.