Receptor Tyrosine Kinase

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RECEPTOR TYROSINE KINASE

Transmits information from a factor outside a cell to the interior of a cell without requiring that factor tocross the cell membrane-Cell surface receptor that has intracellulartyrosine kinase domain-Has a single transmembrane spanning receptorprotein- Largest family of enz linked receptors-monomers initially-p- on tyrosine residue-~50 RTK have been identified-has 1 or 2 polypeptide chains-has intrinsic kinase activityEx: EGF,PDGF, FGF,IGF.

STRUCTURE

Extracellular polypeptide ligand binding domainglycosylated (promotes specific, high affinitybinding of ligand to the receptor)Single pass Trans membrane helix-hydrophobicIntracellular catalytic kinase domain

FUNCTION

Regulates cell growth, differentiation, migration,metabolism, proliferation, apoptosis, andtranscription

MECHANISM:

R (monomer) –L (gf) and R bind ---- R (dimerisation by non covalent binding) and R -Cytoplasmic domainauto p-this Stimulate kinase activity (catalytic domain)- auto p- and p- other molecules.on autop-,receptor recruits CYTOPLASMIC SH2 prt (src homology domain) .thus activates several pathways at thesame time. The transmission of a signal from the membrane to nucleus for activation of transcription can occur bymultiple pathwaysEX: Ras-raf - mapk,PIK3 pathway.Initiated by receptor tyrosine kinase and G-protein-coupled receptors followed by transmission of signalsby proteins that are regulated by kinases and phosphatases. In turn, these activated signalling elementsphosphorylate amino acid residues on downstream signalling proteins. Proto-oncogenes that areimportant cell regulatory genes encodes proteins that function in the signal transduction pathwayscontrolling normal cell proliferation while oncogenes are abnormally expressed or mutated resulting inabnormal cell proliferation and tumour development. The central elements in the MAPK pathway are a family of protein –serine/threonine kinases such as Ras,growth factor receptor binding protein (Grb2),rapidly accelerated fibro sarcoma (Raf),MAP kinase/ERK kinase (MEK),extra cellular signal-regulated kinase (ERK),protein kinase B (PKB/Akt),phosphatidylinositol 3-kinase (PIK3),p21-activated kinase kinase (PAK).

RAS

Ras genes are small (21 to 24 Kda)Monomeric guanine nucleotide binding proteinsFunction in transduction of mitogenic signals froma variety of growth factor receptors. This protein was first identified as the oncogenicproteins of tumour viruses that cause sarcoma inrats.Ras proteins are prototypes of large family of ~50related proteins, called small GTP –bindingproteins.

Ras is synthesized in the cytoplasm and

isdependent upon several posttranslationalmodifications of a C-terminal “CAAX” motif such

asfarnesylation, Proteolysis, to increase itshydrophobicity,

and promote membraneassociation.

RAS ISOFORM : LOCATION, SIGNALLING ANDBIOLOGICAL ACTIVITIES

Ras proto-oncogenes encode four highlyconserved 21 Kda protein (p21

ras)

. They are N-ras(neuroblastoma cell line), H-ras (Harvey murinesarcoma virus), and K-ras (Kirsten

murine sarcomavirus A and B), that are most often

encountered inhuman cancers. Ras has a sequence comprising188–189 amino acids, with a cysteine residue,positioned four amino acids from the C-terminusthat requires addition of a farnesyl group (a 15-carbon terpenoid). They also carry an S-palmitoyl(C

16

fatty acid) group. The C-terminal amino acids(186-189) form a conserved CAAX box required forcorrect posttranslational processing of p21

ras

.Without these groups, the Ras proteins do notassociate with the cell membrane and are found tobe inactive.

ACTIVATION OF RAS PATHWAY

The binding of epidermal growth factor (EGF) induces receptor dimerisation and autophosphorylation (P)on tyrosine residues on the cytoplasmic side of the cell membrane. These phosphotyrosines activates thesmall G-protein Ras by stimulating the exchange of guanosine diphosphate (GDP) for guanosinetriphosphatases (GTP). Tyrosine Phosphorylation of receptors creates a binding site for a small proteincalled Grb2 SH2 domain. Grb2 associated with another small protein called Son of sevenless through abinding domain (SH3) recognises proline-rich regions on Sos.Once activated, this receptor-GRB2-Sos protein complex catalyses the exchange of GTP for GDP resultingin formation of the active Ras-GTP complex. Activation of the Ras GTP complex is then terminated byGTP-hydrolysis, which is stimulated by the interaction of Ras-GTP with GTPase-activating proteins. Thisexchange elicits a conformational change in Ras. This then recruits a kinase called Raf, from the cytosolto the plasma membrane where Raf can be activated by other signals. This ultimately activates apromoter gene within the cell nucleus where transcription of DNA leading to messenger RNA (mRNA)production can then proceed, and these codes for the production of enzymes associated with cell growthand differentiation. Thus Ras proteins are controlled by GTP-GDP binding.

RAS ONCOGENES

The ras oncogenes are not present in normal cells; they are produced in tumour cells as a result of mutation that occurs during tumour development. The Ras oncogenes differ from their proto-oncogenesby point mutation (single amino acid substitution at position Gly12/Gly13 or Gln61). Ras oncogenicactivation by point mutation results in inability to hydrolyse the GTP in presence or absence of GTPase-activating protein (GAP) resulting in continuous expression of Ras favouring malignancy. Mutated Rasgenes are found in about 15% of all human cancers including 25% of lung cancer, 50% of colon cancer,and 90% of pancreatic cancers. Mutation converts normal Ras genes to oncogenes in human cancers andinhibits GTP hydrolysis by the Ras protein. Thus mutated Ras oncogenes maintain the Ras protein continuously in the active GTP-boundconformation. Then, GAP stimulates hydrolysis of bound GTP by normal Ras. Due to decrease inintracellular GTPase activity, the oncogenic Ras proteins remain in the active GTP bound state and driveunregulated cell proliferation. Thus it could be concluded that Ras is not only capable of inducingabnormal growth but also appears to be required for the normal cells proliferation, even in the absenceof growth factors that would be required to activate Ras and signal proliferation of normal cells.Ras ismutated frequently in human cancers. Ras proteins are targeted to plasma membrane by theposttranslational modification by the attachment of lipids to the polypeptide chains. Thus plasma

membrane associated Ras proteins involved in the control of cell growth and differentiation are modifiedand are responsible for the uncontrolled growth of human cancers. Such modifications anchor theproteins to the plasma membrane and interact with the hydrophobic lipid. This type of modification iscalled farnesylation, in which 15-carbon hydrophobic farnesyl isoprenyl tail is added to carboxyl terminusof Ras, catalysed by farnesyl transferase. This step is followed by proteolytic removal of the cysteineresidue at the carboxyl-terminus and methylation (addition of methyl group) of cysteine to the carboxylgroup of the C-terminal cysteine residue.

TARGTING RAS PATHWAY FOR CANCERTHERAPY

Targeting Ras as a therapeutic strategy raisesthe possibility of developing drugs that mightselectively act against cancer cells. Thus, it hasattracted considerable interest as potential drugtargets. Inhibitors of enzyme farnesyl transferaseare found not only to inhibit Ras membranelocalization and function, but also displayconsiderable selectivity in their action againsttumour cells expressing oncogenic Ras proteins.When drugs such as the farnesyl transferaseinhibitors block farnesylation, Ras is unable toanchor to the cell membrane and its function isimpaired.

RAS PATHWAY INHIBITORS

Other inhibitors of Ras are geranylgeranyltransferase-I (GGTase-I), isoprenylcysteinecarboxylmethyltransferase (ICMTase-I), statins,bisphosphonates, small inhibitory RNA (SiRNA),combination of inhibitors such as gefitinib,erlotinib, bevacizumab, lonafarnib are also usedto silence Ras expression in human cancer celllines and the effects of Ras silencing onproliferation, apoptosis, and tumour growth areassessed. The potential of these drugs intreating human cancers that involve in rasoncogenes now awaits evaluation in clinicaltrials.

RAF KINASE

The Raf Serine/ threonine specific protein kinase signalling pathway has been highly conservedthroughout evolution, and activation of the Raf protein kinase is considered to be a primary event in theRas signalling pathway. This Raf signalling pathway promotes cell survival, proliferation and apoptosis. Itwas first identified as oncogenes in retroviruses that are causative vectors in tumours in mice andchicken. There are 3 Raf genes. A-Raf, B-Raf and c-Raf.

A-RAF, B-RAF AND C-RAFA-Raf

(68 Kda) located on Xp11 is the weakest activator of MEK, and can

only activate MEK1 but notMEK2 undergoes localization to the mitochondria which links with the regulation of apoptosis and is overexpressed in urogenetial tissues (Kidney and ovary). So far, no mutations in A-Raf has been found inhuman cancers.Alternatively spliced

B-Raf

(94 Kda) located on 7q32 is the strongest Raf kinase that induces MEK activity that is over expressed in neural, testicular and haemopoietic tissues. Recent studies have shown30 single-site missense activating mutations

within the B-Raf kinase domain in human cancers. This hasprimarily directed to consider B-Raf as a potential therapeutic target. The majority of the somaticmutations

of B-Raf are found in two regions: the glycine-rich loop

of the kinase domain, and within oradjacent to

the activation segment. A Glu for Val substitution at residue

600 (V600E, previouslydesignated V599E) is seen in 90% of B-Raf

mutations, resulting in constitutive kinase activity, which cantransform NIH3T3 cells. B-Raf mutations have been found at inhibitory Akt phosphorylation sites in theCR2

domain. Recent evidence shows that presence of B-Raf mutations may resolve

sensitivity to drugsthat target the ERK pathway at the level

of Raf kinase.

C-Raf

(Raf-1, 74 Kda) is a mitochondrial protein located on 3p25, which undergoes localization to themitochondria. It is ubiquitously expressed in adult tissues. Mutations in

Raf-1 have not been detected inhuman cancers

STRUCTURE AND FUNCTION OF RAF KINASE

Raf protein consists of 3 regions: amino terminus(regulatory domain), activation loop andcarboxyl terminus (catalytic domain). All 3 Raf genes have 3 conserved regions, and severalregulatory phosphorylation sites: CR1 (adjacentto N’), CR2, CR3 (adjacent to C’). The GTP-boundform of ras directly interacts with N-terminalregion of C-Raf. This binding localizes Raf to theplasma membrane. Initial process of Rasactivation requires active GTP binding with Rasbinding domain of Raf CR1 while CR2 rich inproline and threonine residues, is involved inprotein-protein interaction, negative regulationof Raf activity by Akt or protein

kinase Aphosphorylation at serine (S) residue S259 andin localization of Raf and the catalytic domain of

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