Biol220 Cellular Signalling. Non-receptor tyrosine kinases

Transcription

1 Biol220 Cellular Signalling Non-receptor tyrosine kinases

2 The 7TM receptors initiate signal transducton pathways through changes in tertiary structure that are induced by ligand binding. A fundamentally different mechanism is used by a number of other classes of receptor. Here ligand binding leads to changes in quaternary structure specifically the formation of receptor dimers.

3 Human growth hormone (GH) is a monomeric protein of 217 amino acids that forms a compact 4 helix bundle structure. In the young animal GH plays an essential role in general body growth. Secreted from the pituitary gland - release follows a daily rhythm with periodic high amplitude secretory bursts. The hormone is structurally very similar to prolactin which has more specific growth-promoting action on the mammary glands.

4 The growth hormone receptor comprises 638 amino acids, divided into an extracellular domain of 250 amino acids, a single membrane-spanning helix and an intracellular domain of 350 amino acids. In the absence of bound hormone, the receptor exists as a monomer. Growth hormone binds to the extracellular domain of the receptor.

5

6 Unusually, each monomeric hormone binds to TWO receptor molecules, promoting the formation of a receptor dimer. The binding between the hormone and the receptors is highly cooperative; once a hormone has bound to a single receptor molecule, the binding of the second receptor is highly favoured.

7 Dimerization of the extracellular domains of the receptor also brings together the intracellular domains of the receptors.

8 A protein kinase called Janus kinase 2 (JAK2) is bound to each intracellular domain. At the carboxyl terminus is a protein tyrosine kinase domain. Adjacent to this domain is a second region that is homologous to a protein kinase however several key residues have been changed and the function of this domain is not clear. This pair of kinase and kinase-like domains accounts for the name of the protein Janus was the two-headed Roman god of doors and gateways! (alternatively just another kinase!) At the amino terminus is the ERM domain that helps anchor JAK2 to membranes. Between this domain and the kinase domains is an SH2 domain that recognizes peptides containing phosphotyrosine. There are a number of members of the JAK family including JAK1, JAK2, Tyk2 and JAK3.

9 Dimerization of growth hormone receptors brings together the JAK2 proteins associated with each intracellular domain apparently bringing the activation loop of one kinase domain into the active site of the kinase bound to the other receptor which results in cross-phosphorylation. Phosphorylation of tyrosine residues within the activation loop leads to activation of the kinase.

10 When activated by crossphosphorylation, JAK2 can phosphorylate other protein substrates. At least two important proteins are phosphorylated in response to growth hormone action. A regulator of gene expression called STAT5 (STAT signal transducers and activators of transcription) and the growth hormone receptor itself. STAT5 is phosphorylated on a tyrosine residue near the carboxyl terminus of the protein. The resulting phosphotyrosine residue binds to an SH2 domain of another STAT5 molecule.

11 Reciprocal interactions lead to the formation of a stable STAT dimer. The dimerized STAT has a much greater affinity for specific binding sites on DNA than does the monomeric protein and consequently causes regulation of gene expression. There are 6 mammalian STATs and they can form homo or heterodimers after phosphorylation. The phosphorylation of the growth hormone receptor may have several consequences. The phosphorylated receptor may serve as a docking site for JAK2 through its SH2 domain. Also, other proteins may associate with the phosphorylated receptor participating in other signalling pathways.

12 Cytokines The cytokines are soluble proteins involved in cell-cell communication, cell growth regulation, differentiation and cell survival. Interferons (INFs) are involved in responses to viral infections and are mediators of immune responses. Interleukins (ILs) promote proliferation and differentiation of B cells, T cells and mast cells. Erythropoietin (EP) promotes the synthesis of erythrocytes. These cytokines all bind receptors that activate the JAK/STAT signalling pathway.

13 Erythropoietin (EP) The circulating red blood cell mass remains constant. Exposing individuals to high altitude (i.e. low oxygen tension) results in increased erythropoiesis (red blood cell synthesis) whereas hypertransfusion (hyperoxia) results in decreased erythropoiesis. Increase of EP with degree of anaemia in patients with intact kidneys (black triangles) and lack of this increase in patients with renal failure (open squares).

14 Erythropoietin (EP) is produced in the kidney and an intrarenal O 2 sensor is thought to monitor the availability of O 2 and to adjust the rate of EP production accordingly. Plasma EP is a sialoprotein (> 40% carbohydrate, mainly sialic acid) consisting of 165 amino acids. EP affects erythroid progenitors in the bone marrow by binding the EP receptor. The effects of EP are not noticed for 2-3 days after stimulation, due to the time it takes for reticulocytes to mature. EP bound to the extracellular portion of EP-R

15 The role of EP is to enhance proliferation of erythrocyte precursor cells in bone marrow into erythroblasts (cells destined to become erythrocytes) and to stimulate proliferation of newly formed erythroblasts.

16 Role of EP in disease Anaemia results from any number of events that lead to haemorrhage, haemolysis or decreased red blood cell production by bone marrow. Some patients with rheumatoid arthritis, chronic infection or malignancies may become anaemic because of lowered levels of EP. Neuraminidase (a sialase enzyme) inactivates EP and may play a role in producing anaemia. Chronic renal disease can also lead to decreased production of EP. EP has potential therapeutic value in the management of anaemias where the responsive stem cells and erythroid precursors are available but EP plasma levels are low. Commercially produced recombinant human EP is routinely used in the treatment of patients with renal failure.

17 EP and protein tyrosine phosphatases (PTPs) Tyrosine phosphatases have the potential to exert negative influences on the JAK/STAT signalling pathway. One of the best examples of the growth suppressive function of a cytoplasmic PTP involves HCP (haematopoietic cell phosphatase), which is expressed exclusively in haematopoietic cells. Mutations in the gene for HCP cause the motheaten phenotype in mice. These mice have many haemotopoietic and immunological abnormalities. In the absence of HCP there is sustained tyrosine phosphorylation and consequent proliferation of erythrocyte precursor cells. One of the substrates for JAK2 is tyr-429 of EP-R which serves as a docking site for HCP (HCP contains SH2 domains). Once recruited to EP-R, HCP can dephosphorylate JAK2 and downregulate or terminate the signal.

18 Blood doping is the process of artificially increasing the number of red blood cells in the circulation in order to improve athletic performance. In the past this was accomplished by transfusion now outlawed. The same effect can be obtained by injection of recombinant human EP. In the 1998 Tour de France, several team doctors were caught with thousands of doses of EP and ultimately about 50% of the teams withdrew from the race either for cheating or in protest. There is now a urine test that can detect the differences between normal and synthetic EP. Nonetheless blood doping continues to be a problem.

19 Middle-distance runner Rashid Ramzi, (Bahrain) won the 1,500m race at the Beijing Olympics (2008). He was eventually found guilty of doping and was stripped of his gold medal. He and four other athletes tested positive in April in retrospective tests for CERA (continuous erythropoietin receptor activator). The athletes' samples were originally tested at the Beijing Games. They tested negative at the time, but the IOC subsequently reanalyzed the samples when a fully validated test for CERA became available. CERA differs from erythropoietin through the integration of a large polymeric chain that is linked via amide bonds between amino groups and methoxy polyethylene glycol-succinimidyl butanoic acid. Studies in healthy subjects have shown that CERA has a significantly prolonged half-life compared with erythropoietin..

20 Why is erythropoietin dangerous? EP, and transfusion blood doping, is dangerous because of increased blood viscosity. The % of whole blood occupied by red blood cells is referred to as the haematocrit. Above a critical level (it is normally ~ 48% for men and ~ 38% for women), whole blood can sludge and clog capillaries. If this happens in the brain it results in a stroke. In the heart, a heart attack. This has happened to several athletes who have used EP. EP use is especially dangerous to athletes who exercise over prolonged periods. During demanding exercise, as fluid losses mount, water is shifted out of the blood stream (i.e. haematocrit rises). If one is already starting with an artificially elevated haematocrit then it is very easy to enter the sludge zone. Additional dangers of EP include sudden death during sleep, which has killed some 18 pro cyclists in the past fifteen years, and the development of antibodies directed against EP. In this later circumstance the individual develops anemia as a result of the body s reaction against repeated EP injections.

21 Summary In contrast to 7TM receptors that are coupled to G proteins, some other plasma membrane receptors undergo dimerization following ligand binding. Dimerization may lead to activation of an integral receptor tyrosine kinase (eg. EGF receptor) or a non-receptor tyrosine kinase (e.g. GH receptor and JAK). Activation of JAK leads to phosphorylation and dimerization of STAT. The STAT dimer is able to bind DNA and stimulate specific gene expression. The cytokines (e.g. interferons, interleukins and erythropoietin) all operate via the JAK/STAT signalling pathway.