#Protein phosphorylation, kinases, and phosphatases #

1. Protein phosphorylation, kinases
and Phosphatases
Presented by
NATRAJ PREMKUMAR
AD20188502
Graduate Researcher (Ph.D.)
Dept. of Biochemistry
College of Veterinary Medicine.

2. Contents
• Introduction
• Serine/threonine kinases
• Tyrosine kinases
• Mitogen-activated protein kinases
• Phosphatases
• Ubiquitin-proteasome system

3. Introduction
• Protein phosphorylation is an important cellular regulatory mechanism as
many enzymes and receptors are activated/deactivated by phosphorylation
and dephosphorylation events, by means of kinases and phosphatases.
• Virtually all types of extracellular signal, including neurotransmitters,
hormones, light, neurotropic factors and cytokines, produce most of their
diverse physiological effects by regulating phosphorylation of specific
phosphoproteins in their target cells.
• In particular, the protein kinases are responsible for cellular transduction
signaling and their hyperactivity, malfunction or over expression can be
found in several diseases, mostly tumors.
• This reversible mechanism occurs through protein kinases and consists of
the addition of a phosphate group (PO4) to the polar group R of various
amino acids

4. • For phosphorylation to be an effective control mechanism allowing the
activity of an enzyme to be both increased and decreased, the overall
reaction has to be reversible.
• Example : The mechanism of control of glycogen metabolism.

5. The mechanism of phosphorylation regulation consists of kinases,
phosphatases and their substrates phospho-binding proteins

6. Some of the enzymes controlled by phosphorylation/
dephosphorylation and the metabolic pathways in which they are
involved

7. • The addition of phosphate groups to proteins and their removal, are enzyme
catalyzed events.
• The enzymes that catalyze protein phosphorylation are known as protein
kinases, and their reciprocal group of enzymes that carry out
dephosphorylation, called phosphatases.
• However, phosphorylation usually takes place on one of only three amino acids
in the primary sequence of the polypeptide, on either a serine, threonine or
tyrosine ,Although as we shall see there are expectations.
• The two main groups are
 Serine/threonine kinases that add the phosphate to serine and/or threonine.
 Tyrosine kinases that only use tyrosine as acceptors of the phosphate.
• Dephosphorylation is the simple removal of the phosphoryl group from the
amino acid with regeneration of the hydroxyl side chain and release of
orthophosphate.

8. Phosphorylation of serine and tyrosine yields the altered residues
phosphoserine and phosphotyrosine.

9. G- Proteins
Guanine nucleotide binding proteins which act as a Transducer
between a receptor & an effector
Discovered by Alfred Gilman & Martin Rodbell in 1990
Significance:
• 3rd largest family of genes (865)
• Present in almost every organ system

10.  Molecular Switch: On/Off
 Heterotrimerc
α-subunit
β-subunit
γ-subunit
α subunit: specific recognition of
receptors & effectors;
GTP binding site
 βƔ subunit: Membrane localisation by prenylation of Ɣ subunit

11. G Protein Activation
Conformational change in
receptor
α subunit exchange GDP
with GTP
Presence of GEF‟s(Guanine
exchange factors)
Release of GTP bound α
subunit & βƔ dimer

12. In activation
 Activated α subunit is
inactivated by hydrolysis of
GTP to GDP by GAPs
(GTPase Activating
Proteins)
 Rebinds to βƔ complex
 Modulated by
Regulators of G
proteins Signaling (RGSs)
 Acclerate hydrolysis of
GTP & potential drug
targets

13. Serine/Threonine Kinases
cAMP dependent protein
kinases
cGMP dependent protein
kinases
Ca2+ calmodulin dependent
protein kinases

14. cAMP
System
cGMP
System
Phospho-
inositol
System
Tyrosine
Kinase
System
Ligands Epinephrine
Ach
ANP, NO Oxytocin PDGF
Primary
Effector
Adenyl
cyclase
Guanylate
cyclase
Phospho-
lipase C
Receptor
Tyrosine
kinase
Secondary
messenger
cAMP cGMP IP3 & DAG;
Ca2+
-
• Protein kinases differ in their cellular and subcellular distribution, substrate
specificity and regulation.
• The kinases preferentially phosphorylate the amino acids serine or threonine within
polypeptides, and therefore come under the classification of serine /threonine
kinases.
• Encompass a large group of phosphorylating enzymes, including cAMP-dependent
protein kinase, cGMP-dependent protein kinase, Ca2+ - calmodulin dependent protein
kinases.

15. Adenyl Cyclase- cAMP Pathway

16. cAMP-dependent protein kinase (protein kinase A; PKA)
• In multicellular animals virtually all the diverse effects of cAMP are mediated
through protein kinase A (PKA), also called cAMP-dependent protein kinase.
• Another second messenger used in many different cell types is cyclic adenosine
monophosphate (cyclic AMP or cAMP), a small molecule made from ATP.
• In response to signals, an enzyme called adenylyl cyclase converts ATP into cAMP,
removing two phosphates and linking the remaining phosphate to the sugar in a ring
shape.
• Once generated, cAMP can activate an enzyme called protein kinase A (PKA), enabling
it to phosphorylate its targets and pass along the signal.
• Protein kinase A is found in a variety of types of cells, and it has different target
proteins in each. This allows the same cAMP second messenger to produce different
responses in different contexts.
• cAMP signaling is turned off by enzymes called phosphodiesterases, which break the
ring of cAMP and turn it into adenosine monophosphate (AMP).

17. • Inactive PKA is a tetramer
consisting of two regulatory (R)
subunits and two catalytic (C)
subunits.
• Each R subunit has two distinct
cAMP-binding sites; binding of
cAMP to both sites in an R subunit
leads to release of the associated C
subunit, unmasking its catalytic site
and activating its kinase activity.
cAMP has following major targets:
1. cAMP dependent Protein Kinase A (PKA)
2. CREB (cAMP responsive element binding protein)
3. cAMP regulated Guanine nucleotide exchange factors termed EPACs
(Exchange Proteins Activated by cAMP)

18. 2.cAMP Response Element-Binding (CREB)
 Cellular transcription factor

19. 3. Exchange Proteins Activated by cAMP (EPAC)
 cAMP Regulated Guanine
Nucleotide Exchange Factors
 Bind to GDP liganded
GTPase, exchange of GDP
for GTP
 Activation of PKC
 Cell differentiation/proliferation,cytoskeletal
organization, & nuclear transport
 Additional effector system
 Potential target for cancer therapy

20. cGMP-dependent protein kinase (PKG)
• cGMP is also an important cell signaling molecule in cells .The levels of cGMP in cells,
like that of cAMP, can be used to control phosphorylation often via cGMP-dependent
protein kinase or cGK or PKG (protein kinase G).
• PKG shows a much more limited cellular distribution and substrate specificity than
PKA. This reflects the smaller number of second messenger actions of cGMP in the
regulation of cell function.
• The first messenger, nitric oxide, stimulates cGMP production by directly activating
guanylyl cyclases .
• cGMP phosphodiesterases hydrolyze cGMP and are the pharmacological targets of
therapeutic agents such as sildenafil used to treat erectile dysfunction

22. Calcium/calmodulin-dependent protein kinases
(CaM kinases; CaMKs)
• Calcium ions (Ca2+) impact nearly every aspect of cellular life.
• The processes of calcium signaling consist of the molecular and biophysical events that
link external stimuli to the expression of appropriate intracellular responses by means
of increases in cytoplasmic Ca2+.
• The external signal is most commonly a neurotransmitter, hormone or growth factor
but, in the case of excitable cells, the initial chemical stimuli may bring about
membrane excitation, which in turn activates a calcium-signaling pathway.
• CaMKs transfer phosphates from ATP to serine or threonine kinases residues in
proteins in response to increase in concentration of intracellular calcium ions.
• They are important for expression of various genes because after activation, CAMKs
phosphorylate several transcription factors.
• The enzyme exists under physiological conditions as large multimeric complexes of
identical or distinct subunit isoforms of 50–60 kDa.

24. Protein kinase C
• Protein kinase C (PKC) comprises the other major class of Ca2+ -dependent protein kinases and is
activated by Ca2+ in conjunction with DAG and phosphatidylserine .
• PKC exists under physiological conditions as single polypeptide chains of about 80 kDa. Each
polypeptide contains a regulatory domain, which, in the resting state, binds to and inhibits a
catalytic domain.
• For example, they differ in the relative ability of Ca2+ and DAG to activate them: some require
both Ca2+ and DAG, whereas others can be activated by DAG alone, apparently without an
increase in cellular Ca2+ concentrations .

25. AMP-activated protein kinase
• A protein kinase that has recently been shown to be important in a variety of
pathways is 5’ AMP-activated protein kinase (AMPK).
• In the active state it phosphorylates, and inactivates, several metabolic enzymes
involved in the synthesis of fatty acids and cholesterol, and it has also been shown
to be important for insulin signaling, where inhibition of AMPK in the presence of
glucose activates insulin secretion.
• It is expressed in a number of tissues, including the liver, brain, and skeletal
muscle.
• When AMPK phosphorylates acetyl-CoA carboxylase 1 (ACC1) or sterol regulatory
element-binding protein 1c (SREBP1c), it inhibits synthesis of fatty acids,
cholesterol, and triglycerides, and activates fatty acid uptake and β-oxidation.

26. Haem-regulated protein kinase

27. Tyrosine Kinases
Receptor Tyrosine
Kinases
Cytosolic Tyrosine
Kinases

28. • 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 a subclass of protein kinase.
• The phosphate group is attached to the amino acid tyrosine on the protein. Tyrosine
kinases are a subgroup of the larger class of protein kinases that attach phosphate
groups to other amino acids (serine and threonine).
• Phosphorylation of proteins by kinases is an important mechanism in communicating
signals within a cell (signal transduction) and regulating cellular activity, such as cell
division.
• Protein kinases can become mutated, stuck in the "on" position, and cause unregulated
growth of the cell, which is a necessary step for the development of cancer.
• Therefore, kinase inhibitors, such as imatinib, are often effective cancer treatments.
• Most tyrosine kinases have an associated protein tyrosine phosphatase, which removes
the phosphate group.

29. Receptor Tyrosine Kinases
• A second major class of protein kinases including those that add a phosphoryl group to
tyrosine , as opposed to serine or threonine.
• Receptor tyrosine kinases (RTKs) are the high-affinity cell surface receptors for many
polypeptide growth factors, cytokines, and hormones.
• Receptor tyrosine kinases have been shown not only to be key regulators of normal cellular
processes but also to have a critical role in the development and progression of many types
of cancer.
• Mutations in receptor tyrosine kinases leads to activation of a series of signaling cascades
which have numerous effects on protein expression .
• Receptor tyrosine kinases are part of the larger family of protein tyrosine kinases,
encompassing the receptor tyrosine kinase proteins which contain a trans membrane domain,
as well as the non receptor tyrosine kinases which do not possess trans membrane domains.

31. Cytosolic Tyrosine Kinases
• We turn now to a second important class of cell-surface receptors, the cytokine
receptors, whose cytosolic domains are closely associated with a member of a family
of cytosolic protein tyrosine kinases, the JAK kinases.
• A third class of receptors, the receptor tyrosine kinases (RTKs),contain intrinsic
protein tyrosine kinase activity in their cytosolic domains.
• The mechanisms by which cytokine receptors and receptor tyrosine kinases become
activated by ligands are very similar, and there is considerable overlap in the
intracellular signal-transduction pathways triggered by activation of receptors in
both classes.
• In this section, we first describe some similarities in signaling from these two
receptor classes. We then discuss the JAK-STAT pathway, which is initiated mainly
by activation of cytokine receptors.

33. Mitogen-activated protein kinases
• In mammalian cells all receptor tyrosine kinases (RTKs), as well as most cytokine
receptors, appear to utilize a highly conserved signal transduction pathway in which
the signal induced by ligand binding is carried via GRB2 and Sos to Ras, leading to its
activation.
• Activated Ras promotes formation at the membrane of signaling complexes
containing three sequentially acting protein kinases that are associated with a
scaffold protein. This kinase cascade culminates in activation of MAP kinase, a
serine/threonine kinase also known as ERK.
• After trans locating into the nucleus, MAP kinase can phosphorylate many different
proteins, including transcription factors that regulate expression of important cell-
cycle and differentiation-specific proteins.
• Activation of MAP kinase in two different cells can lead to similar or different
cellular responses, as can its activation in the same cell following stimulation by
different hormones.

35. Phosphatases
Serine/Threonine
Phosphatases
Tyrosine
Phosphatases

36. Serine/Threonine Phosphatases
• Protein dephosphorylation is catalyzed by phospho hydrolases called protein
phosphatases.
• Ser/Thr-specific protein phosphatases are regulated partly by their location within the
cell and by specific inhibitor proteins.
• Serine and threonine are amino acids which have similar side-chain compositions that
contain a hydroxyl group and thus can be phosphorylated by enzymes called
serine/threonine protein kinases.
• The addition of the phosphate group can be reversed by enzymes called
serine/threonine phosphatases.
• The addition and removal of phosphate groups regulates many cellular pathways
involved in cell proliferation, programmed cell death(apoptosis), embryonic development,
and cell differentiation.
Here are several known groups with numerous members in each:
PPP1 (α, β, γ1, γ2), PPP2 (formerly 2A), PPP3 (formerly 2b, also known as calcineurin)
PPP2C, PPP4, PPP5, PPP6

37. Tyrosine phosphatases
• Tyrosine phosphatases are a group of enzymes that remove phosphate groups from
phosphorylated tyrosine residues on proteins.
• Protein tyrosine (pTyr) phosphorylation is a common post-translational modification
that can create novel recognition motifs for protein interactions and cellular
localization, affect protein stability, and regulate enzyme activity.
• As a consequence, maintaining an appropriate level of protein tyrosine
phosphorylation is essential for many cellular functions.
• Tyrosine-specific protein phosphatases (PTPase) catalyse the removal of a phosphate
group attached to a tyrosine residue, using a cysteinyl-phosphate enzyme
intermediate.
• These enzymes are key regulatory components in signal transduction pathways (such
as the MAP kinase pathway) and cell cycle control, and are important in the control
of cell growth, proliferation, differentiation, transformation, and synaptic plasticity

39. Ubiquitin-proteasome system
• The ubiquitin–proteasome system (UPS) is a complex mechanism where proteins
are first targeted for degradation by the ubiquitination machinery and then
recognized, unfolded and proteolyzed by the proteasome.
• In autophagy-mediated proteolysis, proteins are degraded by the lysosome.
• Although lysosomal proteolysis was initially considered to be a non-selective
system, it has been shown that chaperones and other cargo-recognition
molecules such as ubiquitin determine the degradation of specific proteins by
the lysosome.
• Therefore, the proteasome and autophagy might be interrelated by using
ubiquitin as a common marker for proteolytic degradation.
• Proteasome inhibition has therefore implications in a number of human diseases
such as cancer, inflammation and ischemic stroke and is an important therapeutic
target.