 A protein kinase is a kinase enzyme that modifies

other proteins by chemically adding phosphate groups

to them (phosphorylation).
 Phosphorylation usually results in a functional change
of the target protein (substrate) by changing
enzyme activity, cellular location, or association with
other proteins.
 About 518 genes in humans encode protein
kinases, making about 1.7% of the human
genome.
 Protein kinases are the fourth largest gene family
in humans

◦ C2H2 zinc finger proteins (3%)

◦ G-protein coupled receptors (2.8%)
◦ Major histocompatibility (MHC) complex
protein family (2.8%)
There are 478 conventional eukaryotic protein
kinases (ePKs) plus 106 pseudogenes
◦ 388 Protein-serine/threonine kinases
◦ 90 Protein-tyrosine kinases
◦ 58 receptor PTKs
◦ 32 Non-receptor PTKs
There are 40 atypical protein kinases (e.g.
EF2K/alpha kinases)
478 + 40 = 518
Defects in protein kinase function are associated
directly with so many diseases.

These enzymes are primary targets for therapeutic

intervention.
Kinases are classified by the amino acids they
phosphorylate.
The two main classes of kinases are;
1) Tyrosine kinases (TKs), which phosphorylate
tyrosine, and
2) Serine-threonine kinases, which phosphorylate
serine or threonine.
 Protein-Serine/threonine
 Protein-Tyrosine
◦ Receptor: ligand binding domain and catalytic site on the
same polypeptide
◦ Non-receptor: catalytic domain separate from the
receptor
 Dual Specificity (both serine/threonine and
tyrosine) also occur
Broad Specificity: have several substrates, e.g.,
PKA
Narrow Specificity: have one or a few substrates,
e.g., pyruvate dehydrogenase kinase with one
substrate
Classified by activator: PKA, PKG, PKC
The best understood member of the protein kinase
superfamily is protein kinase (PKA).

It is also called as cAMP-dependent protein kinase

because it is endogenously activated by cAMP.
1) The first protein kinase to be discovered

2) The first to be sequenced and then cloned

3) The elucidation of its structure provided the first

three dimensional template for this family.
4) It is also the only protein kinase whose excellent
coordinates for transition states are available.
5) As a proto-type for understanding the
macromolecular assembly of protein kinase
complexes.
6) Prototype for thinking about diverse strategies for
designing inhibitors
The protein kinase A holoenzyme is a
heterotetramer composed of two types of subunits:
1) Catalytic Subunit
2) Regulatory Subunit
The catalytic subunit is a globular bi-lobal protein;

1) The highly dynamic small lobe serves as the

binding site for ATP

2) The stable large lobe serves as a framework for

the catalytic machinery and also as a docking
scaffold for substrates or inhibitors.
The R subunit is a highly dynamic and modular
protein that serves as one of the major receptors for
cAMP in eukaryotic cells.

The activity of PKA is regulated by its two

regulatory subunits, which form a dimer that binds
to the two catalytic subunits.
At the N terminus is a dimerization/docking (D/D)
domain that interacts with scaffold proteins,
referred as A-kinase anchoring proteins (AKAPs).

Following this domain is a variable region

containing an inhibitor site that docks to the active-
site cleft of the C subunit.

Two tandem cAMP-binding domains (domain A

and domain B) lie at the C terminus.
The phosphate-binding cassette (PBC) is a helix-
loop region where cAMP binds.

The C subunit is locked in a dormant state in the

absence of cAMP through formation of a
holoenzyme inhibitory complex, where the R
subunit dimer binds to two C subunits.

Binding of cAMP to the R subunit unleashes the

catalytic subunit, thereby allowing phosphorylation
of PKA substrates.
Activation of PKA by cAMP is a highly ordered
process.
In holoenzyme complexes with R subunits
containing both cAMP-binding domains, domain A
is inaccessible until cAMP occupies domain B.
This obligatory activation pathway led to the
designation of domain B as a ‘‘gatekeeper’’ for
domain A.
Agonist stimulation of G-protein-coupled receptors
(GPCRs) activates adenylyl cyclase (AC), which
catalyzes the synthesis of cyclic AMP from ATP.

cAMP binds to the regulatory (R) subunits of

protein kinase A (PKA), which causes a
conformational change that releases the active
catalytic (C) subunit.
The catalytic subunit of PKA can then
phosphorylate nearby substrates. PKA represents
the main intracellular effector of cAMP signaling.

cAMP can also activate nucleotide-gated ion

channels, phosphodiesterases (PDEs) such as
PDE4, and guanine nucleotide-exchange factors
(GEFs) that are known as exchange proteins
activated by cAMP (EPACs)
The two main PKA subtypes are defined by the
identity of their regulatory subunits, RI and RII.

These types are functionally non-redundant; within

these classes are a and b subtypes (RI a, RI b, RII
a, and RII b)
• Most AKAPs contain a recognizable sequence
that forms a binding site for the R subunits.

• The majority of known AKAPs bind specifically

to the RII holoenzyme.

• However, several dual specificity AKAPs, which

bind to both PKA subtypes, have been identified.
Protein kinase A phosphorylates and changes the
activity of following molecules;

1) Enzymes

2) Ion Channels

3) Chromosomal Proteins

4) Transcription Factors
1. Sperm Function
PKA play important roles in sperm function, including;
1) Regulation of motility,
2) Sperm capacitation

2. Cardiac Contractility
PKA phosphorylates numerous substrates that may
influence contractility in cardiac myocytes, including;
1) The L-type Ca2+ channel,
2) The ryanodine receptor,
3) Phospholamban, and
4) Troponin I
3. Cancer
PKA type I is associated with growth and proliferation
whereas PKA type II is associated with increased
differentiation and decreased proliferation.

4. Diabetes
 Binding of GLP-1 and GIP to their receptors couples
to activation of adenylate cyclase.

 Intracellular
cAMP levels are elevated, leading to
activation of PKA and cAMP-regulated guanine
nucleotide exchange factor II (cAMP-GEFII).
o The cAMP-pathway is a well described repressor
of macrophage activation and can therefore
suppress inflammatory responses.
o Much of this effect is PKA-mediated through;
1) Inhibition of Toll-like-receptors

2) Inhibition of phagocytic receptors and

3) Production of reactive oxygen species

 Protein kinase B or Akt is a serine/threonine kinase,
which in mammals comprises of three highly
homologous members known as PKB alpha (Akt1),
PKB beta (Akt2), and PKB gamma (Akt3).

 They also plays a key role in multiple cellular

processes such as glucose metabolism, apoptosis, cell
proliferation, transcription and cell migration.
 Akt1 is involved in cellular survival pathways, by
inhibiting apoptotic processes.

 Akt2 is an important signaling molecule in the insulin

signaling pathways. It is required to induce glucose
transport.

 The role of Akt3 is less clear, though it appears to be

predominantly expressed in the brain. It has been
reported that mice lacking Akt3 have small brains.
 It is stimulated by binding of an extracellular ligand
to a receptor tyrosine kinase (RTK) in the plasma
membrane, causing receptor dimerization and cross-
phosphorylation of tyrosine residues in the
intracellular domains.
 The regulatory subunit p85 binds to phosphorylated
tyrosine residues on the activated receptor via its Src
homology 2 (SH2) domain. It then recruits the
catalytic subunit p110 to form the fully active PI3K
enzyme.
 Alternatively, adaptor molecule Grb2 binds to
phospho-YXN motifs of the RTK and recruits p85
via Grb2-associated binding (GAB) scaffold
protein.
 Activated PI3K catalyses the addition of phosphate
groups to the 3'-OH position the inositol ring of
phosphoinositides (PtdIns), producing three lipid
products, PI(3)P, PI(4,5)P2 and PI(3,4,5)P3.
 These phosphorylated lipids are anchored to the
plasma membrane, where they can directly bind
intracellular proteins.
 Akt resides in the cytosol in an inactive
conformation, until the cell is stimulated and it
translocates to the plasma membrane.

 Although PI3K is the major mode of Akt activation,

other tyrosine or serine/threonine kinases have been
shown to activate Akt directly, in response to growth
factors, inflammation or DNA damage.
1. Cell Cycle
Akt is known to play a role in the cell cycle. Under
various circumstances, activation of Akt was shown
to overcome cell cycle arrest in G1 and G2 phases so
activated Akt may enable proliferation and survival
of cells.
2. Angiogenesis
Akt1 has also been implicated in angiogenesis and
tumor development. Although deficiency of Akt1 in
mice inhibited physiological angiogenesis but it
 enhances the pathological angiogenesis and tumor
growth associated with matrix abnormalities in
skin and blood vessels. Akt also contributes to
angiogenesis by activating endothelial nitric oxide
synthase (eNOS), which increases production of
nitric oxide (NO).
3. Role in Schizophrenia
The PI3K/PKB pathway is usually down-regulated
in schizophrenic patients, causing atrophy of
neurons and decrease in dendritic branching.
 Because of the Akt functions above, Akt inhibitors
may treat cancers such as neuroblastoma. Some Akt
inhibitors have undergone clinical trials. In 2007
VQD-002 had a phase I trial. In 2010 Perifosine
reached phase II. but it failed phase III in 2012.
 Protein kinase C, commonly abbreviated to PKC is
a family of protein kinase enzymes that are involved
in controlling the function of other proteins through
the phosphorylation of hydroxyl groups (OH) of
serine and threonine amino acid residues on
these proteins, or a member of this family.
Protein kinase C (PKC) family members regulate
numerous cellular responses including ,
 gene expression
 protein secretion
 cell proliferation
 inflammatory response
 Growth factors
 Hormones
 Drugs (Membrane receptor ligands)
 Domain structure of protein kinase C (PKC) isoforms. Top:
PKCs have a conserved kinase domain (depicted in teal)
and more variable regulatory domains.

 All PKC regulatory domains have a pseudosubstrate motif

(shown in green) NH2 terminal to the C1 domain (shown in
pink). Tandem C1 domains are the molecular sensors of
phorbol 12-myristate 13-acetate (PMA)/diacylglycerol
(DAG) in cPKC and nPKC isoforms, whereas the single
aPKC C1 domain does not bind DAG/PMA.

 The C2 domains (in yellow) function as calcium-dependent

phospholipid binding modules in cPKCs. nPKC C2
domains do not bind calcium; the PKCδ-C2-like domain is
a phosphotyrosine interaction module. PKC isoform
variable regions are shown in gray.
 PKC

◦ C refers to calcium
◦ DAG and phospholipid were also described as
necessary for the activation of this enzyme.
◦ DAG requires phospholipase C for its activity

 It is paradoxical to have PKCs that are independent

of Ca2+ and DAG
 The activation of PKC is preceded by a number of
steps, originating from the binding of an extracellular
ligand that activates a G-protein on the cytosolic side
of the plasma membrane.
 The G-protein, using GTP as an energy source, then
activates PKC via the phosphatidylinositol
bisphosphate (PIP2) intermediate, which is shown as
the DAG/IP3 complex.
 PDGF receptor
 EGF receptor
 Insulin receptor
 Transferrin receptor
 Ribosomal protein S6
 Raf
PKC has been associated with vascular alterations
such as increase in,
 permeability
 contractility
 extracellular matrix synthesis
 cell growth and apoptosis
 angiogenesis,
 leukocyte adhesion
 cytokine activation and inhibition.
 Development of pathologies

Large vessel Small vessel

Atherosclerosis Retinopathy

Cardiomyopathy Nephropathy

Neuropathy
PKC isozymes play critical role in,

 cellproliferation
 survival
 invasion
 migration
 apoptosis
 angiogenesis
Among PKC isozymes,

 PKC𝛼, 𝛽, 𝜀, and 𝛿 have been the most broadly

studied isozymes in relation to cancer.

 This may be associated with their ubiquitous

expression in many tissues .
• Colon cancer
• Breast cancer
• Chronic myeloid leukemia
• Bladder cancer
• Gastric cancer
• Gastointestinal stromal tumor (GIST)
• Hepatocellular carcinoma
• Glioma
• Myeloid and lymphatic leukemia
• Ovarian cancer
• Prostrate cancer….
 Several recent studies have reported that a PKCs may
also act as novel therapeutic targets for cancer
treatment,
e.g., PKC𝜄 in,
 lung,
 ovarian
 colon cancer
1. Calphostin C potent inhibitor of PKC
2. Ruboxisataurin may potentially be beneficial in
peripheral Diabetic retinopathy
3. Chelerythrine ,a natural PKC inhibitor
4. Staurosporine to induce apoptosis
5. Bryostatin 1 can act as a PKC inhibitor; It was
investigated for cancer.
It is serine/threonine kinases that is present in a
variety of eukaryotes ranging from the unicellular
organism Paramecium to humans.

The PKG family is commonly involved when

cGMP signaling is mediated by NO.
cGMP is a second messenger generated from GTP
either by soluble guanylyl cyclase (sGC) or
particulate guanylyl cyclases (pGCs).

The sGC is activated by nitric oxide (NO) or

carbon monoxide, whereas the pGCs bind a family
of natriuretic peptides (NPs).
• It is found in all types of smooth muscle cells
(SMCs) including vascular SMCs and in
platelets.

• Lower levels are present in vascular

endothelium and cardiomyocytes.

• The enzyme is also expressed in fibroblasts,

certain types of renal cells and leukocytes, and
in specific regions of the nervous system.
Stimulation of receptors that raise intracellular cyclic
GMP concentrations leads to the activation of the
cyclic GMP-dependent protein kinase (PKG).

PKG phosphorylates some of the same substrates as

PKA and some that are PKG-specific.

In some tissues, PKG can also be activated by cAMP.

Unlike the heterothermic (R2C2) structure of the
PKA holoenzyme, the catalytic domain and cyclic
nucleotide-binding domains of PKG are expressed
as a single polypeptide, which dimerizes to form
the PKG holoenzyme.
PKG exists in two homologous forms, PKG-I and
PKG-II.

PKG-I has an acetylated N terminus, is associated

with the cytoplasm and has two isoforms (Iα and
Iβ) that arise from alternate splicing.

PKG-II has a myristylated N terminus, is

membrane-associated and can be localized by
PKG-anchoring proteins.
On Smooth Muscles
NO and ANP stimulate cGMP synthesis in vascular
SMCs and relax small arteries and arterioles
resulting in a decreased blood pressure.

NO/NP/cGMP signaling is involved in the

development of vasculoproliferative disorders,
such as restenosis and atherosclerosis.
Platelets generate cGMP only by the soluble, NO-
activated guanylyl cyclase, degrade cGMP via PDE 2
and 5, and contain cGMP effector systems consisting of
PKG and cGMP-regulated PDEs.

Elevation of platelet cGMP is associated with platelet

inhibition.
PKG mediates the negative inotropic effect of
NO/cGMP.

Stimulation of cGMP synthesis by ANP/GC-A or

NO inhibits cardiomyocyte hypertrophy.
Blood pressure is increased by activation of the renin-
angiotensin aldosterone system, which stimulates
vasoconstriction and inhibits natriuresis/diuresis.
These effects are counteracted by regulators that signal
through cGMP such as ANP, urodilatin, uroguanylin,
and locally produced NO.
The therapeutic potential of cGMP-elevating drugs
has been documented by the clinical success of
NO-generating drugs for the treatment of angina
pectoris and congestive heart failure.

PDE 5 inhibitors are approved for the treatment of

erectile dysfunction and pulmonary hypertension.
 Receptor
◦ Insulin
◦ Epidermal Growth Factor
◦ Platelet Derived Growth Factor
 Non-receptor
◦ Src protein kinase
◦ Abl and Bcr-Abl
◦ Jak (Janus kinase [two catalytic regions]) or
whimsically, just another kinase
The EGFR kinase is unusual among the family of
receptor tyrosine kinases in that it does not require
phosphorylation of the activation segment for full
activity.

The EGFR family consists of four members, EGFR

(ErbB1), HER2 (ErbB2, HER2/neu), HER3
(ErbB3), and HER4 (ErbB4).
 Head and neck squamous cell carcinomas (>90%
associated with ErbB overexpression)
 Bladder
 Breast
 Kidney
 Non-small cell lung
 Prostate cancers