Protein kinase conformational changes

Approximately one-third of cellular proteins contain phosphate and are subject to covalent modification by phosphorylation and dephosphorylation reactions. This reversible phosphorylation of proteins causes conformational changes in the protein that dramatically alters their properties, e.g. from an active to an inactive enzyme, or vice versa. The sites of protein phosphorylation are those amino acid residues that contain hydroxyl groups, most commonly serine but also tyrosine and threonine (Fig. 27.2) (Chapter 31). Phosphorylation uses protein kinase and dephosphorylation uses protein phosphatase. The importance of reversible protein phosphorylation to the living cell is emphasised by the fact that protein kinases and protein phosphatases comprise approximately 5% of the proteins encoded by the human genome. Current research is discovering abnormalities of protein phosphorylation that are associated with diseases, notably type 2 diabetes meUitus (T2DM) and cancer. In the future, the discovery of drugs that modify protein phosphorylation/dephosphorylation promises new therapies for the treatment of these diseases. 

Conformational changes in a protein kinase are important for cell cycle regulation... 

Figure 13.32 Regulation of the catalytic activity of members of the Src family of tyrosine kinases, (a) The inactive form based on structure determinations. Helix aC is in a position and orientation where the catalytically important Glu residue is facing away from the active site. The activation segment has a conformation that through steric contacts blocks the catalytically competent positioning of helix aC. (b) A hypothetical active conformation based on comparisons with the active forms of other similar protein kinases. The linker region is released from SH3, and the activation segment changes its structure to allow helix aC to move and bring the Glu residue into the active site in contact with an important Lys residue.

The current understanding on activation of Tec kinases fits into a two-step model. In the first step an intramolecular interaction between the SH3 domain and aproline-rich region in the TH domain is disrupted by binding ofthe PH domain to phosphoinositides, G protein subunits, or the FERM domain of Fak. These interactions lead to conformational changes of Tec and translocation to the cytoplasmic membrane where, in a second step, Src kinases phosphorylate a conserved tyrosine residue in the catalytic domain thereby increasing Tec kinase activity. Autophosphorylation of a tyrosine residue in the SH3 domain further prevents the inhibitory intramolecular interaction resulting in a robust Tec kinase activation. 

So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca +/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca + and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca + in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca +/calmodulin protein kinase inhibitor. However, whereas depolarisation... 

The reaction of X with S must be fast and reversible, close to if not at equilibrium with concentration of S. It can be that there is an intermediate step in which X binds to a protein kinase (a protein which phosphorylates other proteins mostly at histidine residues in bacteria) using phosphate transferred from ATP. It then gives XP which is the transcription factor, where concentration of S still decides the extent of phosphorylation. No change occurs in DNA itself. Here equilibrium is avoided as dephosphorylation involves a phosphatase, though changes must be relatively quick since, for example, cell cycling and division depend on these steps, which must be completed in minutes. We have noted that such mechanical trigger-proteins as transcription factors are usually based on a-helical backbones common to all manner of such adaptive conformational responses (Section 4.11). 

Because phosphate groups are negatively charged, phosphorylation of a protein alters its charge, which can then alter the conformation of the protein and ultimately its functional activity. A change in the state of protein phosphorylation can be achieved physiologically through increases or decreases in the activity of either protein kinases or protein phosphatases. Examples of each of these mechanisms occur in the nervous system, often in concert with one another, to elicit complex temporal patterns of protein phosphorylation. 

---