2009 Group 4 Project

From CellBiology

'"Tropomyosin-receptor-Kinase (Trk)"'

Introduction

The tropomyosin-receptor kinase (trk) belongs to the family of tyrosine-protein kinase receptors (TK), which was discovered as a proto-oncogene that consisted of seven exons of ‘non-muscle tropomyosin’ gene fused to the cytoplasmic domains of a novel ‘tyrosine-kinase’ gene. Trks are commonly associated with cell survival, proliferation, neurite growth including axons & dendrites as well as regulatory function of ion channels and neurotransmitter receptors in immature neural cells. In addition, binding of ligands to trks can affect growth, plasticity and synaptic strength of neuronal junctions in the adult nervous system (ref), although it is notable that Ligand binding does not itself illicit an action potential event. Three trk genes have been identified in mammals: trkA (proto-oncogene), which serves as an NGF receptor, trkB and trkC, that each was subsequently identified due to their high homology to trkA(ref).

Structure

  • Trk receptors are made of an extracellular segment that binds polypeptide ligands, transmembrane helix, and cytoplasmic segment where the tyrosine kinase catalytic activity takes place.[1]
  • TrK receptors act as dimeric transmembrane proteins. Distally, three cysteine-rich motiffs and two Leucine rich regions form a conserved NGF binding region which is common to all TrK's near the amino terminus.[1]
  • Additionally, the common binding region is flanked by a pair of extracellular immunoglobulin-like domains located proximally to the cell membrane. The juxtamembrane complex contains a variable amino acid sequence suggested to determine ligand bindng affinity and specificity and may bind directly to some ligands; The localization of these peptides appears to be non-specific in tertiary NGF-TrKA complex structures however their conformation within this complex appears to indicate direct participation in complex binding.
  • Further NGF binding site affinity is generated through collaboration with the p75 protein/neurotrophin receptor (p75NTR). Furthermore, p75NTR regulates NT-3-TrkA and NT-4/5-TrkB mediated receptor activation by blocking Neurotropin ligation by circumnavigating the prequisite of these neurotropins for receptor activation.

Function

TrK receptors achieve their function primarily via neuroptrophin signaling. There are three types of trk receptors, namely trkA, trkB and trkC. These receptors have different binding affinity to certain types of neurotrophins. Hence, the signalling by these distinct receptors will result in various cellular functions.Trk receptors regulate cell survival, proliferation, axon and dendrite growth and the expression and activity of ion channels and neurotransmitter receptors.[2] TrK also regulate synaptic strength and plasticity in adult nervous system. As a family of tyrosine kinase receptors, TrK receptors are activated and regulated in a similar fashion. TrK receptor signaling is involved in G proteins activation and pathways regulated by MAP kinase, PI3K and PLC.[3]

  • Trk A

Trk A also known as Neurotrophic tyrosine kinase, receptor, type 1 is a high affinity receptor for nerve growth factor (NGF). Binding of NGF to TrK A leads to the recruitment of proteins that interact with specific phosphotyrosine residues in the cytoplasmic domains of Trk receptor A. Such interactions activate signaling pathways, such as the Ras, PI3K, and PLC pathways and result in activation of gene expression, neuronal growth and proliferation.[2][3]

  • Trk B

Trk B also known as Neurotrophic tyrosine kinase, receptor, type 2 is a high affinity receptor for brain derived growth factor (BDNF) and neurotrophin-4 (NT4). Trk B are consisted of 3 isoforms in human central nervous system. The isoform (TK+) is a typical tyrosine kinase receptor, and transduce the BDNF signal via the common signaling pathways such as Ras-ERK, PI3K, and PLCγ. The other two isoforms, TKT1 and TKT2 are distinct from the typical kinase receptors as the C-terminal sequences are T1 and T2 specific.[2][3]

  • Trk C

Trk B also known as Neurotrophic tyrosine kinase, receptor, type 3 is a high affinity receptor for neurotrophin-3 (NT3). Binding of NT3 on Trk C triggers the neuronal growth from neural stem cells in a process known as neurogenesis. NT3 is particularly important in proprioceptive neuron growth.[3]

Maintenance and Proliferation of undifferentiated Cell Lines

Dynamic Process

NGF binding to ligation region facilitates dimerisation of Trk Receptor monomers into the signal tranductive unit- activating the kinase unit. Phosphorylation of the autoregulatory loop tyrosines of the cytoplasmic domain further activates the kinase. Phosphorylation of an additional seven tyrosines within this region promotes signalling by providing docking sides for adaptor proteins which regulate signalling-cascade couples.

*Incomplete* Trk dynamics

Mechanism of Trk Activation via multiple Signalling pathways

Tropomyosine receptor kinases (Trk) enhance neuronal cell growth and differentiation of neurites via the activation of various signalling pathways. PKC, PLCy1, Ras/MAPK and PI3 are the four well known pathways, which exhibit this activity. The latter process involves the activation of transcription factors that will bind onto a specific target gene, thus trigerring neuronal cell division and/ differentiation via production of specific proteins that inhibit apoptosis.


PLC-y1 Pathway

Simplified PLC γ1 Pathway. Full sized animation

Phospholipase C, comes in many forms and plays and important role in the signal transduction for many processes, including, proliferation, differentiation and motility. Its main function is to hydrolyze phosphatidylinositoldi phosphate (PIP2) into diacylglycerol (DAG) and inositoltriphosphate (IP3). [4] This occurs by the activation of the membrane bound receptor by a number of growth factors, cytokines and immunoglobins. [5]

DAG is necessary for the further activation of the PKC and it remains bound to the cell membrane. IP3, a water soluble protein , in turn is released into the cytoplasm and leads to the release of Ca2+ reserves from the endoplasmic reticulum. This increase of calcium ions actives the classic isoforms of [Protein Kinase C|Protein kinase C]. [6]


PKC Pathway

See also: Protein Kinase C

Simplified Animation of PKC pathway. Full size animation

Protein Kinase C (PKC) is a family of nucleotide-independent, Ca2+-dependent serine kinases. [7]. At least 11 isozymes have been identified, [8] most PKC isozymes are ubiquitous and multiple members of the family can be coexpressed within the same cell leading to complex pathways. However, the basic signal transduction pathway involves the allosteric activation of PKC by the intracellular messengers diacylglycerol and Calcium ions. The inactive PKC is mainly found in the cytosol. Upon stimulation the PKC proteins translocate to the cell membrane. This translocation is the result of a cascade initiated by the binding of a number of extracellular ligands, such as, growth factors, hormones, and neurotransmitters to a G protein-coupled receptor (GPRC) found on the plasma membrane. This in turn activates a stimulating G protein which in turn activates phospholipase C (PLC). [9] A G protein is then activated, which then activates phospholipase C (PLC). The PLC cleaves phosphoinositol-4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol-1.,4,5-triphosphate (IP3). [10] The IP3, couples to Ca2+ channels found on the endoplasmic reticulum releasing Ca2+ reserves. The increased concentration of calcium ions bind to inactive PKC molecules and then translocates to the plasma membrane. Here is binds to DAG giving rise to the active PKC enzyme. [7] The PKC family plays a major role in cellular signal transduction. [11]Their main roles consist in the regulation of cell proliferation, differentiation, survival and apoptosis. [12]


Ras-MAPK (mitogen-activated protein kinase) Pathway

Activation of Ras-MAPK signalling pathway is predominantly essential for promoting neuronal cell growth and differentiation. Several pathways derive from Trk receptors for activation of Ras molecules, which begin by formation of ligand-bound mitogen (BDNF or NGF) at an extracellular domain of the trk, causing receptor dimerization. This subsequently will lead to phosphorylation of the intracellular parts of the receptors, which activates Guanine Exchange factors (GEFs), such as sos, Grb2 and Shc. GEFs trigger the exchange of GDP bound to inactive Ras to GTP, resulting Ras to activate. The presence of activated Ras molecules, stimulate signaling via a major downstream pathway known as MAP kinase (mitogen-activated protein kinase)-although activated Ras, would be also capable of triggering several other downstream pathways such as PLC-y1 and PI3-kinases. (Figure..)


Mitogen-activated protein kinase (MAPK) pathway involves a series of signalling cascade. Raf is the first component of MAPK, activated by Ras-GTP on the plasma membrane. Raf then phosphorylates MEK1/2,which in turn activates the ERK1/2 by phosphorylation. ERK1/2 kinase would further phosphorylate a variety of downstream targets. These pathways may include phosphorylation of MAP 2-kinase by MAP 3-kinase on its serine and threonine amino-acid residues. Further phosphorylation of MAP- kinase by MAP 2-kinase on its serine and tyrosine amino-acid residues subsequently yields activated MAP-kinase. This results in activation of transcription factors that will lead to changes in gene expression. (Click here for well illustration of MAP-kinase pathway on You-tube) Nonetheless, aberrant Ras-MAPK signaling has been identified to be a possible factor responsible for the uncontrolled proliferation and malignancy (See “Cancer Biotherapy”, current research).


PI3K (Phosphatidylinositol 3-kinase) Pathway

See also: Phosphatidylinositol 3-kinase

The involvement of PI3K in intracellular signaling

PI3K are a family of intracellular transducer enzymes, and are involved in different signaling pathways and the control of key functions of the cells.[13] PI3K is also involved in motor neuronal diseases due to its role in tyropomyosine kinase receptors.PI3Ks have three subclasses based on their structure and substrate specificity. They are all composed of 85 kd and 110 kd subunits. The ability of PI3K to activate a number of signal proteins including some oncoproteins determines its significance in regulation of cell functions such as growth and survival, aging and malignant transformation.[13] Pl3K is also involved in the control of both malignant cell resistance to mutating agents and the sensitivity of malignant tumors to chemotherapy and radiotherapy.[14] PI3K has an array of main effectors which include the mitogen-transducing signal proteins such as protein kinase C, phosphoinositide-dependent kinases and mitogen activated protein kinases. They are activated either via their interaction with lipid products of PI3K or through PI3K-dependent phosphorylation of proteins.[15] PI3K is activated by number of growth factor receptors with intrinsic and tyrosine kinase activity. Nerve growth factor (NGF) activates PI3K in TRK to regulate cell functions.[16]

Current Research

Cancer biotherapy via targeting ‘Ras / Ras-MAP-kinase’ signalling pathway

Recent studies have hypothesized that receptors responsible for Ras activation would be effective target for treating human cancers (ref). Based on this hypothesis, since 1995, therapies utilizing the inhibition of Ras activation have aroused a major interest. Farnesylation is a process by which small G-proteins like Ras are activated and adhered to the cell membrane. In a recent paper (Sebti & Hamilton 2000), farnesyl transferase inhibitors were used in order to block the farnesylation of Ras proteins. The results revealed promising hopes in animal models but unfortunately have not been effective in human cancer treatment. The major downstream target of Ras-MAP-Kinase (MAPK) has also been taken as a subject for drug design. Sebolt and his colleagues showed that MAPK-inhibitors are effective in cancer therapy using mouse models (ref). It has been also suggested that Inhibition of the Ras-MAPK signaling pathway can serve as a potential therapy for NF1 in humans (ref).

Trk receptors can behave as signalling endosomes

In a recent publication, a hypothesized model was explained in which active Trk receptors are internalised into signaling endosomes during signalling transduction process (ref). The experiment that led to this understanding involved local application of NGF to axons and soma (cell bodies) of neuronal cells. It was shown that both ERK1/2 and ERK5 signaling cascades were activated. Surprisingly, application of NGF to neuronal cells at distance resulted only in activation of ERK5 pathway and NOT ERK1/2. It was then understood that activation of ERK5 in the cells somas required receptor internalisation due to inhibition by thermo-sensitive dynamin. Further investigation was conducted by local application of specific Trk-inhibitor (K252a) at either the distal axon or cell body, which blockaded the activation of ERK5 in the cell soma in the presence of NGF. Therefore, the hypothesized model in which active Trk receptors are internalised into signalling endosomes is inevitable. Nonetheless, the mechanism through which distance affects the specificity of signalling would be the future topic of investigation.

The interplay between miRNA and Trks (tropomyosine-receptor-kinases)

Under construction

Timeline

under construction


Glossary

  • Autophosphorylation is the term used to define the phosphorylation of a kinase protein catalysed by its own enzymatic activity.
  • GTPases are a large family of enzymes that can bind and hydrolyse guanosine triphosphate(GTP).
  • NGF-Nerve growth Factor.
  • Exons-Expressed regions of eukaryotic genes.
  • Ras- small G-proteins that belong to the superfamily of monomeric GTPases. They are involved in receptor-mediated signal transduction pathways.
  • BDNF- Brain-derived neurotrophic growth factor is a protein that promotes activation of tropomyosine receptor kinases (trk).
  • NGF- Nerve growth factor is a protein that promotes activation of tropomyosine receptor kinases (trk).
  • Phosphorylation- A process by which a phosphate group is added to a protein that is required for its cellular activation.
  • ERK- Extracellular regulated kinase.
  • Apoptosis- Programmed cell death.
  • Farnesyl transferase inhibitors- a class of experimental cancer drug blocks farnesylation of Ras proteins.
  • Neurofibromatosis (NF)- an inherited disorder that causes production of benign tumours on nerve tissues. There are two types of NF; NF1 and NF2. NF1 is the most common disease amongst infants, also known as Recklinghausen disease.
  • Dynamin- GTPase enzyme responsible for endocytosis in eukaryotic cells.


References

  1. 1.0 1.1 EJ, Reichardt LF. “Trk receptors: roles in neuronal signal transduction”. Annu Rev Biochem. 2003;72:609-42. Retrieved 2 May 2009 from Pubmed
  2. 2.0 2.1 2.2 Pyle AD, Lock LF, Donovan PJ. Neurotrophins mediate human embryonic stem cell survival. Nat Biotechnology 1996;24:344–50. Retrieved 13 May 2009 from [1]
  3. 3.0 3.1 3.2 3.3 Melamed et al., A Novel Lymphocyte Singnaling Defect: trk A Mutation in the Syndrome of Congenital Insensitivity to Pain and Anhidrosis (CIPA). Journal of Clinical Immunology, 2004;24:(4)441-448. Retrieved on 13 May 2009 from [2] Cite error: Invalid <ref> tag; name "me" defined multiple times with different content Cite error: Invalid <ref> tag; name "me" defined multiple times with different content Cite error: Invalid <ref> tag; name "me" defined multiple times with different content
  4. PhD, Hershel Raff. Physiology Secrets. Philadelphia: Hanley & Belfus, 2002.
  5. Mankidy, R., Hastings, J., & Thackeray, J. (2003). Distinct Phospholipase C-#-Dependent Signaling Pathways in the Drosophila Eye and Wing Are Revealed by a New small wing Allele. Genetics Society of America. 164(2): 553–563.
  6. Bioengineering; Research from University of Tokyo broadens understanding of bioengineering. (2009,May). Medical Devices & Surgical Technology Week,70.
  7. 7.0 7.1 Takai, Y., Kishimoto, A., Inoue, M., & Nishizuka, Y. (1977). Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J. Biol. Chem., 252(21), 7603-7609. [3]
  8. Acs, P., Wang, Q., Bogi, K., Marquez, A., Lorenzo, P., Biro, T., Szallai, Z., Mushinski, F., & Blue, P. (1997). Both the Catalytic and Regulatory Domains of Protein Kinase C Chimeras Modulate the Proliferative Properties of NIH 3T3 Cells. J Biol Chem., 272(45), 28793-28799. Retrieved May 15, 2009, from Pubmed
  9. Dekker, L (2004). Protein Kinase C (Molecular Biology Intelligence Unit). New York: Springer.
  10. Signal Transduction Resource. (n.d.). Retrieved May 15, 2009, from Promega
  11. Steinberg, S. (2008). Structural Basis of Protein Kinase C Isoform Function. Physiol. Rev., 88, 1341-1378. Retrieved October 5, 2009, from Pubmed
  12. D Breitkreutz, L Braiman-Wiksman, N Daum, M F Denning, T Tennenbaum. (2007). Protein kinase C family: On the crossroads of cell signaling in skin and tumor epithelium. Journal of Cancer Research & Clinical Oncology, 133(11), 793-808. Retrieved May 16, 2009, from Pubmed
  13. 13.0 13.1 Krasilnikov MA. Phoshatidylinositol-3 kinase dependent pathways: the role in control of cell growth, survival, and malignant transformation. Biochemistry. Review 2000;65(1):59-67. Retrieved May 16, 2009, from Pubmed Cite error: Invalid <ref> tag; name "kr" defined multiple times with different content
  14. Carpenter et al., A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity. Molecular Cell Biology. 1993;13(3):1657-1665. Retrieved May 16, 2009, from Pubmed
  15. Kapeller R, and Cantley LC. Phosphatidylinositol 3-kinase. Bioessays. 1994;16(8):565-576. Retrieved May 16, 2009, from [4]
  16. Stephens LR, Jackson TR, and Hawkins PT. Agonist-stimulated synthesis of phosphatidylinosital(3,4,5)-triphosphate: a new intracellular signaling system? Biochim Biophys Acta 1993;1179(1):27-75. Retrieved May 16, 2009, from Pubmed



2009 Group Projects

--Mark Hill 14:02, 19 March 2009 (EST) Please leave these links to all group projects at the bottom of your project page.

Group 1 Meiosis | Group 2 Cell Death - Apoptosis | Group 3 Cell Division | Group 4 Trk Receptors | Group 5 The Cell Cycle | Group 6 Golgi Apparatus | Group 7 Mitochondria | Group 8 Cell Death - Necrosis | Group 9 Nucleus | Group 10 Cell Shape