1. Revised curriculum (1) December 16 (Tuesday) Second messengers
(2) December 18 (Thursday) Protein kinases and cancers
(3) December 23 (Tuesday) Protein phosphatases
and diseases
(4) December 25 (Thursday) Literature study I
(5) December 30 (Tuesday) Literature study II
Tzu-Ching Meng
ext 6140 or 6141

2. Assigned reading for literature study I

3. Assigned reading for literature study II

4. Basic concept of cell signaling and second messengers
Lecture I
Basic concept of cell signaling and
second messengers
Reference:
Lodish et al., Molecular Cell Biology 6th edition (2007),
Chapters 15 and 16
December 16, 2008

5. Cell as information processing unit
Unit A
Unit B
Unit C
signals
System
System
Unit
Signal
society
person
language
computer
circuit
electrical
organism
cell
chemical
Cell
Input signal
Output signal

6. General principles of signaling by cell-surface receptors
Synthesis and (2) release of signaling
molecules by the signaling cells;
(3) Transport of the signal to the target cells;
(4) Binding of the signal by a specific
receptor protein;
(5) Initial of intracellular signal transduction
pathways;
(6) Specific changes in cellular functions;
(7) Inactivation of the receptor;
(8) Removal of signaling molecules

7. Signaling molecules can act locally or at a distance

12. Two modes of intracellular signaling transmission
Low molecular weight messenger substances– second messengers
Protein-protein interaction
Overview of major classes of cell surface receptors
2nd messengers

13. Relationship between ligand and receptor—
G protein coupled receptors

14. General structure of G protein coupled receptor

15. Operational model for ligand-induced activation of effector protein
associated with G protein coupled receptors

17. Overview of major classes of cell surface receptors
Protein-protein interaction

18. Relationship between ligand and receptor—
Receptors that activate intracellular enzymes

19. Overview of major classes of cell surface receptors
Protein-protein interaction
Proteolytic process

20. Relationship between ligand and receptor—
Receptors that trigger intracellular proteolysis pathways

21. Relationship between ligand and receptor—
Receptors that function as ion channel upon ligand binding

22. A distinct class of receptors—
Receptors that are located intracellularly, but ligands may be
generated either extracellularly or intracellularly

23. Characteristics of second messengers:
Diffusible signaling molecules;
Rapidly produced/activated and rapidly degraded/inactivated;
May be stored intracellular in certain organelles;
Can be produced in a location-specific manner.
Two types of second messengers:
Hydrophilic– cAMP, cGMP, inositol phosphates (IPs), Ca2+, NO;
Hydrophobic– diacylglycerol (DAG),
phosphatidyl inositol phosphates (PIPs).

24. Four common 2nd messengers

25. Mammalian adenylyl cyclase is a membrane-bound enzyme with two catalytic
domains and two integral membrane domains, each of which contains 6
transmembrane a-helices.

26. 3-D structure of Gsa-GTP complexed with
two catalytic domains of adenylyl cyclase
One subunit of trimeric G protein

27. Hormone-induced activation or inhibition of
adenylyl cyclase in adipose cells Gs
stimulation Gi
inhibition

28. Typical amplification of an external signal downstream from a receptor
Extracellular stimulus
Effector
Intracellular 2nd messenger
Effector

29. The membrane A kinase-associated protein (mAKAP) anchors both PKA and
cAMP phosphodiesterase (PDE) to the nuclear membrane, forming a feedback
loop that provides close local control of the cAMP level
Basal level of PDE activity keeps [cAMP] low;
Activation of receptor causes an increase in [cAMP]binding of cAMP to PKA;
Activated PKA (C) phosphorylates and activates PDE, driving [cAMP] low;
Dephosphorylation of PDE returns the complex to the resting state.

30. Synthesis of DAG and IP3 from membrane-bound phosphatidylinositol
Key enzyme 1
Key enzyme 2
Key enzyme 3
All membrane-bound

31. IP3/DAG pathway triggered by ligand binding to GPCRs for elevation of
Cytosolic Ca2+ and activation of PKC

32. Second messenger nitric oxide (NO) is produced
by intracellular NO Syntheases (NOSs)
NADP
NADPH
nNOS (NOS1)
L-citrulline
iNOS (NOS2)
L-arginine
eNOS (NOS3) O2
NO.
NO synthases
在細胞中存在三種NOS isoforms, 主要由第一次被發現的位置或者是本身活性上的特性來命名. 分為nNOS, 最初是在neuron cell中發現,也稱為NOSI,另一個為INOS,i為inducible的意思,也稱為NOSII,第三類則是eNOS,是在endothelial cell中被發現,也稱作NOSIII.這3種NOS isoforms目前已知存在在許多的tissue或cell type中.
NOS enzyme執行的功能是藉由催化L-arginine以及在其它cofactor的幫助之下,例如NADPH.O2,將L-arginine轉化成L-citrulline以及NO.最初NO的產生被認為是在bacteria中可以發生,後來在mammalian係統中可藉由NOS來產生NO,因此也突顯了NO的重要性.
NO is generated in most cell types by NO synthases.
nNOS: neuronal NOS
iNOS: inducible NOS
eNOS: endothelial NOS

33. Guanylyl cyclase is a typical intracellular receptor of NO
NOS
NO/cGMP-
dependent
pathways
L-arginine NO +
citrulline
guanylyl cyclase
GTP
cGMP
cGMP dependent的方式是NO與guanylyl cyclase heme group上的鐵離子作用,造成結構上的改變而活化gyanylyl cyclase,具活性的gyanylyl cyclase可以將GTP催化產生cGMP, cGMP在分別由以下三種不同路徑,phosphodiesterases(PDE)以及 protein kinase G還有一些ion channel的影響來調控cellular effects.
我們來看一下cGMP所影響到的層面有哪些.
在這裡我們可以看到,S-nitrosylation是對蛋白上的cysteine接上NO group的修飾作用,另外NO也可以對蛋白進行其他種形式的修飾,例如在蛋白上的tyrosine接上NO2 group修飾,稱為nitration.目前的了解,S-nitrosylation是在細胞中確實可以進行的蛋白修飾,而對於nitration這樣的蛋白修飾還有所保留.而我們的研究主要就是針對S-nitrosylation進行探討,
cGMP-regulated
Phosphodiesterases
(PDE)
Protein kinase G
cGMP-regulated
Ion channels
Smooth muscle relaxation, platelet inhibition ,
cell growth and differentiation

34. Regulation of contractility of arterial smooth muscle by NO and cGMP

35. ROS= Reactive Oxygen Species

36. Reactive oxygen species (ROS) function as second messengers in cells
UV,
Ionizing
Irradiation
Peptide Growth
Factors
Ligands for
GPCRs
Cytokines
H2O2
Nox
ROS
ROS
ROS ?
Activation of
Tyr(P) signaling
Transformation
Signaling
Mitogenic Signaling

37. Assigned paper for the Literature Study I
Huang et al (2007) Positive regulation of Itk PH domain function by
soluble IP4. Science, 316, p. 886
Reference article:
Irvine R. (2007) Cell signaling: the art of the soluble. Science, 316, p. 845