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ADVANCED CELL BIOLOGY//
MOLECULAR BIOLOGY OF CELLS
ā¢Tuesday 25.11.08 (8:40-10:00)
ā¢SEC-
http://lifesci.rutgers.edu/~denhardt/course/cellmolbiol.htm
ā¢Dr. Guy Werlen
ā¢Dept. of Cell Biology & Neuroscience
ā¢Nelson Bio. Labs., B
ā¢werlen@biology.rutgers.edu
ā¢Office hours, Friday 3:30pm-5:00 pm
Molecular Cell Biology
6th Edition
Chapter 16:
Cell signaling II: Signal pathways that control gene activity
Lodish ā¢ Berk ā¢ Kaiser ā¢ Krieger ā¢ Scott ā¢ Bretscher ā¢Ploegh ā¢ Matsudaira
- 16.2 Cytokine Receptors and the Jak/STAT pathway.
- 16.3 Receptor tyrosine kinases.
- 16.4 Activation of Ras and the MAP kinase Pathways.
- 16.5 Phosphoinositides as Signal Transducers.
- 16.7 Pathways that involve Signal-Induced Protein Cleavage.
- Some slides (signaling in T cells) may also refer to chapter 24 .5,
Pathways that control gene expression
Distinct intracellular mechanisms transduce signals downstream of each of the eight major classes of cell-surface receptors. Direct activation of cytosolic transcription factors following TGF and cytokine receptor activation (a, b). Alternatively, receptor stimulation leads to the activation of cyosolic protein kinases that, by translocating into the nucleus activate nuclear transcription factors (c, d). In other pathways, active transcription factors are released from multiprotein complexes (e,f) or by proteolysis (g, h). Some receptor classes can trigger more than one intracellular pathway.
Cytokine receptors
Many cytokine receptors are members of the hematopoietin-receptor superfamily, named after the first of its members to be defined. This large superfamily also known as the class I cytokine receptors, is divided into 3 subsets on the basis of differences in sequence and structure. These class of receptors are heteromers in which the Ī± chain often defines ligand specificity, while the Ī² & Ī³ chains confer intracellular signaling function. A smaller number comprises the class II cytokine receptor superfamily of which many bind interferons or interferon- like cytokines. Members of the tumor necrosis factor receptor (TNFR) family form an other superfamily of cytokine receptors. The ligand for this class of receptors act as trimers and may be associated with the cell membrane rather than being secreted. The chemokine superfamily belongs to the very large family of G-protein coupled receptors (GPCR).
Cytokine receptors belong to superfamilies of receptor proteins, each with a distinctive structure. Each superfamily member is a variant with distinct ligand specificity, performing a particular function on the cell that expresses it.
Cytokines control ontogeny, growth and activation of many hematopoietic cells
Red blood cells White blood cells (f.ex. T lymphocytes)
Erythropoietin controls maturation of erythroid progenitor cells. Erythroid progenitor cells (CFU-E) are derived from hematopoietic stem cells. In the absence of Erythropoietin (Epo), CFU-E cells undergo apoptosis. Binding of Epo to its specific receptor (EpoR) on the surface of CFU-E induces gene transcription of anti-apoptotic proteins as well as proteins involved in cell division. Upon terminal differentiation, the EpoR is lost and mature red blood cells are no longer capable to respond to Epo.
Interleukin-2 (class I cytokine) controls proliferation of naĆÆve T cells during an immune response and ensures clonal expansion of a particular T cell type. (cf. chapter 25.4)
Colony forming units erythroid
Deficiency or mutations of cytokine receptors have dramatic consequences
Targeted deletion of EpoR, the receptor for erythropoietin leads to anemia and death of e13 old embryos
Deletion or mutation the common Ī³ chain of class I cytokine receptors induces a severe combined Immunodeficiency Syndrome. T cells fail to develop because many cytokines that share the Ī³-chain (IL-2, IL-4, IL-7, IL-9 & IL-15) can not activate their target cells.
Activation of JAK kinases is the proximal event that transduces cytokine signals intracellularly
The cytocsolic domain of cytokine receptors tightly and irreversibly associates with separate JAK kinases. 1) in the absence of ligand, the receptors form dimers but the JAK kinases are poorly active because a structural ālipā protects the catalytic site. Ligand binding causes a conformational change that brings the active domains of the JAKs together. Ensues cross-phosphorylation on tyrosine residues located on the JAKās activation lip (2). This causes the lip to move out of the kinase catalytic site, thus increasing the ability of ATP to bind and the activated kinase can now phosphorylates several tyrosine residues in the receptorās cytosolic domain (3). The resulting phosphorylated tyrosines function as docking sites for inactive STAT transcription factors and other proteins that contain SH2 or PTB domains.
A tyrosine phosphorylated cytokine R can recruit many different SH2 containing proteins that will transduce the signal to distinct signaling pathways
Four major pathways can transduce signals downstream of activated and phosphorylated EpoR-JAK complexes. Each pathway ultimately regulates the transcription of sets of different genes.
Long term cytokine deactivation by protein degradation via SOCS and the proteasome
SOCS (suppressor of cytokine signaling) binds to phosphorylated tyrosine residues of the cytokine receptor, thus blocking the association of STATs or other SH2 containing signal transducers with the cytokine receptor. SOCS recruits the E3 ubiquitin ligase, which targets the receptor for degradation by the proteasome.
Receptor tyrosine kinases (RTK)
Ligand binding to a monomeric RTK induces a conformational change and dimerization
Dimerization of 2 identical ligand-bound RTK monomers occurs primarily through interactions of the activated loop segments
Intrinsic kinase activity regulates RTK signaling
Classical RTKs have intrinsic tyrosine kinase activity. Dimerization activates the tyrosine kinases, which cross-phosphorylate each other on tyrosine residues of the activation lip. This causes the lip to move out of the catalytic site of the respective kinase and alows ATP binding. The activated kinase phosphorylates additional Ys on the cytosolic domain of the RTK.
The HER receptors bind epidermal growth factor (EGF) or related growth factors
HER1 homodimerizes, while HER3 lacks a functional kinase domain and can signal only when dimerized to HER2. HER2 does not directly bind a ligand; it needs to heterodimerize with an activated HER1, HER3 or HER4. HER2 overexpression in breast epithelial cells leads to breast cancer (in 25% of breast cancer patients).
EGF= epidermal growth factor HB-EGF= heparin binding-EGF TGFa= tumor-derived growth factor NRG1= neuregulin NRG2+ neuregulin
The T cell receptor (TCR) a atypical RTK
The TCR is a heterodimer of a Ī±- and a Ī²-chain that lack intrinsic tyrosine kinase activity. Several CD3 proteins are constitutively attached to the TCR. The CD3 isoforms control TCR expression, as well as signal transduction downstream of the TCR. A ligand for the TCR must at the same time bind to the CD4 (or CD8) coreceptor. This activates the tyrosine kinase Lck that is associated to CD4 (or CD8). Activated Lck will phosphorylate specific tyrosine residues in the ITAMs (immunoreceptor tyrosine-based activation motifs) of the CD3 isophorms. Phosphorylation of ITAMs will attract the SH2 domain containing tyrosine kinase, ZAP-70. ZAP- bound to CD30 can be phosphorylated and activated by Lck. For ref. see chapter 24.5 (figure 24-31).
Ī±-CPM mutant TCR
Ī²āchain
(Partially functional)
Ī±āchain
Ī±āCPM
An evolutionarily conserved domain of the TCR Ī± -chain transduces survival signals during thymocyte differentiation
b
Ī± Ī± Werlen et al, 2003, Science 299 , 1859- (original ref within this paper).
Mutation of the evolutionary conserved connecting peptide motif of the TCR Ī±- chain (Ī±āCPM) blocks thymocyte differentiation at the CD4 +CD8+^ double positive stage. This block induces a sever immunodeficiency as no T cells are found in the periphery (lymph node) of TCR transgenic mice that carry the Ī±āCPM mutation.
Scaffold proteins (āadaptorsā) that contain SH2 or PTB domains associate to phosphorylated tyrosine residues of RTKs
Cytosolic proteins that carry SH2- or PTB domains (in purple and respectively maroon) can bind to specific phosphorylated tyrosine residues of RTK. These proteins are the phosphorylated on tyrosines by associated or intrinsic receptor tyrosine kinases. Proteins that contain multiple tyrosine residues serve as scaffolds to nucleate many distinct SH2 or PTB containing signalin molecules.
Genetic studies in Drosphila have identified key signaling molecules and mechanisms of the RTK/MAPK pathways
a) Several ommatidia constitute the eye of Drosophila. B) An ommatidia is a tubular structure formed by 8 photoreceptors. C) Ommatidia with the sevenless mutation lack the R photoreceptor cell.
a)The R8 cell in each ommatidia expresses a surface Protein, Boss, that binds to the RTK, Sev expressed on the surface of the neighboring R7 precursor cell. b) This interaction activates a gene program that triggers the differentiation of the R7 precursor cell into a fully functional R7 photoreceptor cell. In the sevenless mutation the Boss-Sev interaction does not take place, consequently the R7 precursor cell stays immature. C) Differentiation of R7 can be rescued by a constitutively active Ras protein.
Larval development
The binding of Sos to inactive Ras induces GDP/GTP exchange
Sos pries Ras open by displacing the Switch I region, which allows GDP to diffuse out of the active pocket of Ras. GTP replaces GDP and the ensuing conformational change displaces Sos and promotes Ras interaction with downstream effectors.
The MAPK signaling pathways
- Ras is activated by exchange of GTP for GDP. 2) The new conformation of Ras allows its binding to, and activation of the Serine/threonine kinase, Raf. 3) The inactivation of Ras by GTP hydrolysis, allows further activation of Raf. 4) Raf phosphorylates a residue in the activation lip of the dual kinase, MEK. 5) Activated MEK, phosphorylates a tyrosine residue in the activation lip of ERK, which induces a conformational change of the lip and the exposure of a threonine residue that is also phosphorylated by MEK, thereby fully activating ERK. 6) ERK dimerizes and translocates to the nucleus.
MAPK ERK1/
JNK1/2/3 p38 (Ī±/Ī²/Ī³/Ī“)
positive selection TH1/TH2 commitment negative selection
Activation
TCR/CD
GTPases Ras Rac/Cdc
MAPKKKK
MAPKKK Raf-1 MEKK-1/4,MLK,ASK1,TAK
MAPKK
(PAK)?
MEK1/2 MKK4/7 MKK3/
ERK, JNK and p38 are the most ubiquitous members of the MAPK family, they are regulated by a canonical module comprising a MAPKKK, a MAPKK and the MAPK. Each of the upstream components are specific to the ERK, JNK or p38 pathway. Each MAPK pathway is linked to a RTK , such as the TCR via a small G protein of the Ras or Rac families. In T cell development, ERK regulates positive selection and differentiation of developing thymocytes, while p38 controls negative selection and death. JNK is mediating TH1 or TH commitment and T cell activation. Werlen et al, 2003, Science 299 , 1859-1863.
Mutation of the Ī± CPM specifically abrogates the activation of the ERK cascade, which is required to transduce a differentiation signal in CD4+CD8+ DP thymocytes
Erk p
Time (^) Time
Time
positive selection ligand
negative selection ligand
Time
Ī± CPM
mutant TCR
High affinity ligand-stimulation induces a fast en transient ERK activation that peaks before maximal JNK and p38 activation. This leads to thymocyte negative selection and death. In contrary low affinity ligands induce a slow and sustained ERK activation that leads to positive selection and thymocyte differentiation. The Ī±CPM mutation of the TCR blocks thymocyte differentiation by specifically abrogating ERK activation. Modified from Werlen et al, 2003, Science 299 , 1859-1863.
MAPKs activation in PC12 pheochromocytoma cells
Proliferation
Time
ERK
activation
Differentiation
NGF
EGF
Modification of activation kinetics is a mechanism by which signal specificity can be achieved. In neuronal cells, EGF triggers a fast and transient ERK activation that leads to cell proliferation. In contrast NGF stimulation induces a slow and sustained ERK activation that results in cell Differentiation.
MAPKs phosphorylate transcription factors and promote gene expression
Each of the MAPK family member can activate specific as well as overlapping transcription factors. ERK activates directly the transcription factor, TCF (ternary complex factor), while SRF is activated via the kinase p90RSK. JNK phosphorylates Jun and thus regulates the activity of the transcription factor, AP-1.
MAPK transduce mating and osmoregulatory signals in yeast
While the organization of MAPK cascades is similar from yeast to man, they are not connected to RTKs in yeast. A GPCR activates the Fus3 pathway and regulates the transcription of genes that are required for mating. Fus3 is a MAPK homologue. Hog1, a distinct MAPK is part of the osmoregulatory pathway and controls the transcription of genes required for osmotic homeostasis.
Protein cleavage is the main signaling regulatory mechanism in the Notch and APP pathways
Notch is cleaved twice upon binding of delta to Notch
Amyloid precursor protein (APP) is also cleaved twice. Cleavage of the extracellular domain by Ī²-Secretase instead of Ī±-Secretase generates the AĪ²42 peptides that form the large amyloid plaques found in Alzheimer patients.
Signal transduction: āthe more you learn, the less it becomes clearā or the complexity of transducing accurately a signal that will induce a specific cell response
Multiple signaling molecules and mechanisms transduce a signal and contribute to a specific cell response.
āGenealogicā tree of human kinases. Deduced from sequence homologies found in the human genome.
