1.
By
Ingole Charan U.(M.tech Part I)
School Of Biochemical Engineering IIT(BHU)
varanasi (u.p)
Characterizations and
Application of Tyrosine Kinase
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Introduction
 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
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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
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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
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Structure5

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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
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 …

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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.
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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.
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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.
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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.
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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.
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Classification of RTK12
 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

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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
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Kinetic study of Tyrosine kinase
(Bruton's tyrosine kinase)
 Two substrate kinetic analysis demonstrates that BTK employs a sequential, or ternary
complex, kinetic mechanism.
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Application
 The Role of Tyrosine Kinase Activity in Endocytosis, Compartmentation, and down
regulation of the Epidermal Growth Factor Receptor
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Application
 Tyrosine kinases as targets for anticancer agents
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Application
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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
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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.
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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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Refrences
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5. Carpenter G, King LJr, Cohen S. Epidermal growth factor stimulates phosphorylation in membrane preparations in
vitro. Nature. 1978;276:409–410. [PubMed]
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7.Nestler, E. J. & Greengard, P. (1983) Nature (London) 305,583-588.
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