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5.3.3 Phosphorylation of proteins as a means of regulating activity

Phosphorylation is an important mechanism for regulating the activity of many proteins, either switching on or switching off some activity of the protein.

  • What protein that we have already discussed is both positively and negatively regulated by phosphorylation?

  • Src kinase activity is switched on by dephosphorylation of Tyr 527 and phosphorylation of Tyr 416. Dephosphorylation of Tyr 416 and phosphorylation of Tyr 527 together switch off the kinase activity.

In a reaction catalysed by a specific protein kinase, the terminal (γ) phosphate group on an ATP molecule is transferred to a hydroxyl group on an amino acid side-chain (Ser, Thr or Tyr) in the target protein (Figure 33). Removal of the phosphate group is catalysed by a phosphatase. The energy required to drive the cycle of phosphorylation and dephosphorylation derives from the hydrolysis of the ATP.

Figure 33 Protein phosphorylation. (a) A phosphate group is transferred from ATP to a hydroxyl group on the protein by a kinase and can be removed by a phosphatase. (b) Phosphorylation can occur on Ser, Thr or Tyr residues.

The cycle of phosphorylation and dephosphorylation can be very rapid, making the activity of the protein exquisitely sensitive to regulation in this way. Frequently, the kinases and phosphatases that catalyse these switches are themselves regulated, either by allosteric regulators or by phosphorylation (as in the case of Src kinase). This kind of cascade of activation and inactivation permits amplification and integration of upstream signals, as well as feedback regulation by downstream components.

How does phosphorylation of a protein affect its activity? Addition of a phosphate group at a crucial residue can change the conformation of a protein or alter the interactions of the protein with substrates or other molecules. There are several ways in which phosphorylation-induced change can happen:

  • The phosphate group may prevent binding of a substrate or ligand. Being strongly negatively charged, the phosphate may disrupt electrostatic interactions between a protein and its ligand. Alternatively, it may block ligand-binding by steric hindrance.

  • Phosphorylation may cause a dramatic change in the conformation of the protein, as in the case of Src, where the activation loop changes to an open conformation, allowing the substrate to bind.

  • The phosphorylated residue in the context of the protein may be recognised by another protein. Some adaptor proteins recognise specific phosphorylated motifs and ‘recruit’ the protein to a protein complex where it may be a substrate in a further reaction. For example, the ‘14–3–3 proteins’ (named, for historical reasons, according to their chromatographic properties), which regulate certain protein kinases, recognise specific motifs containing phosphoserine residues.

  • Which protein domains have you come across that recognise phosphorylated amino acid residues?

  • SH2 and PTB domains bind to specific motifs containing phosphorylated tyrosine residues (Table 5).

There are very many different protein kinases in eukaryotic cells and many share a common structure for their kinase domain. Variations in amino acid sequence and higher-order structure account for their substrate specificity. Though less numerous than kinases, there are also many phosphatases in eukaryotic cells. Some phosphatases are highly substrate-specific, acting on only one or two phosphoproteins, but there are others that can act on a broad range of substrates. In the latter, regulatory domains serve to target the enzyme activity to particular substrates.

Regulation by phosphorylation is a particularly common mechanism in intracellular signalling. However, other proteins that are not signalling molecules are also regulated in the same way, notably some enzymes in metabolic pathways. An example is pyruvate dehydrogenase, which catalyses the oxidation of pyruvate, the endproduct of glycolysis, to give acetyl CoA and CO2. Acetyl CoA then enters the citric acid cycle. Pyruvate dehydrogenase is not actually a single enzyme but is an example of a multienzyme complex (see Section 6.5 ) and comprises three different enzymes. One of these enzymes, pyruvate decarboxylase, is inactivated by phosphorylation of a specific Ser residue. Dephosphorylation reactivates the enzyme. The kinase that catalyses the phosphorylation of pyruvate decarboxylase is subject to allosteric regulation by a number of small molecules, including acetyl CoA, pyruvate and ADP, as indicated in Figure 34.

Figure 34 Regulation of pyruvate decarboxylase by phosphorylation. The phosphorylated enzyme is inactive. The kinase that phosphorylates pyruvate decarboxylase is positively regulated by acetyl CoA and NAD.2H and negatively regulated by pyruvate and ADP.

Acetyl CoA is a positive allosteric regulator of pyruvate decarboxylase kinase which, in turn, phosphorylates and hence inactivates pyruvate carboxylase. In this way, acetyl CoA inhibits its own synthesis.

  • Of what kind of regulation is this an example?

  • Feedback inhibition.


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