1

By Ingole Charan U.(M.tech Part I)

School Of Biochemical Engineering IIT(BHU) varanasi (u.p)

Characterizations and Application of Tyrosine Kinase

2Introduction

Tyrosine kinases are important mediators of the signaling cascade, determining key roles in

diverse biological processes like growth, differentiation, metabolism and apoptosis in response

to external and internal stimuli.

A tyrosine kinase is an enzyme that can transfer a phosphate

 group from ATP to a protein in a cell. It functions as an “on”

or "off" switch in many cellular functions.

Tyrosine kinases are implicated in several steps of neoplastic

development and progression. 

Tyrosine kinases represent a major portion of all oncoprotein

that play a transforming role in a plethora of cancers.

Structural Classification of Proteins (SCOP)• Class: Alpha and beta proteins (a+b)• Fold: SH2-like• Superfamily: SH2 domain• Family: SH2 domain• Protein Domain: Tyrosine-protein kinase

3 Introduction cont. The identification and development of therapeutic agents for disease states that are

linked to abnormal activation of tyrosine kinases due to enhanced expression, mutation or autocrine stimulation leading to abnormal downstream oncogenic signaling have taken a center stage as a potent target for cancer therapy.

The discovery that SRC oncogene having a transforming non receptor tyrosine kinase activity , and the finding of EGFR, the first receptor tyrosine kinase paved the way to the understanding of the role and significance of tyrosine kinase in cancer

4 General Characteristic The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes.Up to

30% of all human proteins may be modified by kinase activity.

Protein Tyrosine Kinase generally made up of Transmembrane Glycoprotein.

Kinetics. The Km of RPK(Bpk) for ATP was 11.6 ,µM . The Km for MAPKK was 0.8 µM.

Substrate Specificity. The stoichiometry of phosphorylation of MAPKK by ptk(BPK)was 1.67 mol of phosphate per mol of MAPKK.

Kinase properties: Km = 10.6; turnover number = 30.fig

the second-order rate constant, kcat/Km, for the phosphorylation of Kemptide(substrate) by PK is

pH sensitive. A plot of this parameter as a function of pH is bell-shaped with the acidic limb (pKa ) 6)

being ascribed to the ionization of a general-base catalyst.

Rate-Determining Steps in Protein Kinases

5 Structure

6 Biochemical mechanism of action of tyrosine kinase

Schematic representation of the mode of action of tyrosine kinase. PK represents protein kinase and PP stands for protein phosphatase

…

7 Mechanism of Tyrosine Kinase Receptors Tyrosine kinase receptors are a family of receptors with a similar structure. They

each have a tyrosine kinase domain (which phosphorylates proteins on tyrosine residues), a hormone binding domain, and a carboxyl terminal segment with multiple tyrosines for autophosphorylation. When hormone binds to the extracellular domain the receptors aggregate.

8 Mechanism of Tyrosine Kinase Receptors When the receptors aggregate, the tyrosine kinase domains phosphorylate the C terminal tyrosine

residues.

This phosphorylation produces binding sites for proteins with SH2 domains. GRB2 is one of these proteins. GRB2, with

SOS bound to it, then binds to the receptor

complex. This causes the activation of SOS.

9 Mechanism of Tyrosine Kinase Receptors SOS is a guanyl nucleotide-release protein (GNRP). When this is activated, it causes certain G proteins

to release GDP and exchange it for GTP. Ras is one of these proteins. When ras has GTP bound to it, it becomes active.

Activated ras then causes the activation of a cellular kinase called raf-1.

10 Mechanism of Tyrosine Kinase Receptors Raf-1 kinase then phosphorylates another cellular kinase called MEK. This cause the activation of MEK.

Activated MEK then phosphorylates another protein kinase called MAPK causing its activation. This series of phosphylating

activations is called a kinase cascade.

It results in amplification of the signal.

11 Mechanism of Tyrosine Kinase Receptors Among the final targets of the kinase cascade are transcriptions factors (fos and jun showed here). Phosphorylation of these proteins causes them to become active and bind to the DNA, causing changes in gene transcription.

12 Classification of RTK Receptor tyrosine kinases (RTKs)-The RTK family includes the receptors for insulin

and for many growth factors such as

Epidermal growth factor (EGF)

Fibroblast growth factor(FGF)

Platelet-derived growth factor (PDGF)

Vascular endothelial growth factor(VEGF)

Nerve growth factor (NGF)

Nonreceptor tyrosine kinases (NRTKs)

Src

Janus kinases (Jaks)

Abl

13 Kinetic study of Tyrosine kinase(Bruton's tyrosine kinase)

Bruton's tyrosine kinase (abbreviated Btk or BTK) is a type of kinase enzyme implicated in the primary immunodeficiency disease X-linked a gammaglobulinemia (Bruton's a gammaglobulinemia)

BTK Western Blot Analysis

Two-substrate Analysis- The entire data set of velocity values was then globally fit to equations describing either a ternary complex (i.e. sequential) or a Ping-Pong enzymatic mechanism. The equation for a ternary complex mechanism is given by,

Vmax [ATP] [S1]

V = ----------------------------------------------------------------------

[ATP][S1]+ [ATP] Km, S1+ [S1] Km, ATP + Km, ATPKa, S1

14Kinetic study of Tyrosine kinase(Bruton's tyrosine kinase)

Two substrate kinetic analysis demonstrates that BTK employs a sequential, or ternary complex, kinetic mechanism.

15Application

The Role of Tyrosine Kinase Activity in Endocytosis, Compartmentation, and down regulation of the Epidermal Growth Factor Receptor

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17 Application Tyrosine kinases as targets for anticancer agents

18Application

19 Application

Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction

Protein-tyrosine kinases (PTKs; EC 2.7.1.112) are believed to play an important role(s) in the metabolism of the cell, most probably as components of signal-transduction pathways.

Indirect evidence in support of this presumption is found in the frequent identification of members of the PI7K family as cellular receptors for certain growth factors and as products of the oncogenes of many of the acutely transforming retroviruses

20 Application

Application of tyrosine kinase inhibitors as a promising targeting treatment for myeloproliferative neoplasms 

The potential use of tyrosine kinase inhibitors in severe asthma.

Kinase targets and inhibitors for the treatment of airway inflammatory diseases: the next generation of drugs for severe asthma and COPD?

A Protein Tyrosine Kinase In the Interferon α/β Signaling Pathway.

21 Conclusion

protein kinases are key regulators of cell processes. Comparison of the structures of protein

kinase domains, both alone and in complexes, allows generalizations to be made about the

mechanisms that regulate protein kinase activation.

Protein kinases in the active state adopt a catalytically competent conformation upon

binding of both the ATP and peptide substrates that has led to an understanding of the

catalytic mechanism. Docking sites remote from the catalytic site are a key feature of several

substrate recognition complexes. Mechanisms for kinase activation through

phosphorylation, additional domains or subunits, by scaffolding proteins and by kinase

dimerization are discussed.

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Thank you !!!

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