C2006/F2402 '07 OUTLINE OF LECTURE #15
Dr. Deborah Mowshowitz, Columbia University, New York, NY.
Last update 03/20/2007 09:13 AM . (Corrections, mostly trivial typos, marked in blue.)
I. Signaling, cont.; Significance of RTK's (Receptor Tyrosine Kinases)
A. Review: What are the 4 basic types of receptors?
1. Steroid Receptors -- Internal (intracellular) receptors for lipid soluble hormones (steroids & TH)
2. GPLRs (or GPCRs) -- Cell surface (transmembrane) receptors linked to G proteins (also called G Protein Coupled Receptors or GPCRs)
3. RTKs -- Cell surface (transmembrane) receptors that are -- or are linked to -- tyrosine kinases.
4. Channels -- some transmembrane receptors are ion channels. These will be ignored for now but discussed at length when we get to nerves & muscles. The most famous example is the nicotinic acetyl choline receptor -- the receptor involved in nerve-muscle signaling.
B. What is special about RTKs? RTK's are representative of a different type of cell surface receptor involved in signaling. (Different from GPCR's -- see table below for details.) Signal on outside binds to receptor, and activation of proteins, often TF's, happens inside. Often no second messenger. How does the binding of a ligand on the outside of the cell activate proteins inside? What's the path of 'signal transduction?' That's topic II today.
C. Role in Development
1. Many embryonic inductions use RTK's.
2. Important ligands for RTK's = FGF's (fibroblast growth factors) = family of related proteins including EGF; act as paracrines (&/or autocrines)
3. An example: how Optic Vesicle induces surface ectoderm to become lens.
- Optic vesicle secretes FGF8 (& BMP4, etc.) = signal molecules = soluble proteins
- Overlying ectoderm makes receptors (RTK's) for FGF8 (under instructions from pax6).
- Binding of FGF8 to TK receptor → signaling pathway → activation of TF's → proteins (including new TF's) for differentiation into lens.
4. Today: Details of the 'signaling pathway?' How does a TK receptor (RTK) cause activation of TF's? See below.
D. Role in Cancer & Regulation of the Cell Cycle
1. RTK's control the normal cell cycle
2. Many cancer cells have mutations affecting RTK's or their targets (proteins normally activated by RTK's).
3. Two major types of mutations responsible -- RTK targets are always 'on' as a result of one of the following:
a. Stuck accelerator (stimulation uncontrolled) -- RTK, its ligands, or targets are either overproduced or stuck in active form
b. Brake failure (inhibition fails) -- Inhibitors of RTK or its targets are missing or inactive.
II. How do RTK's Work?
A. Important Properties of Receptor TKs
1. Receptor is usually a single pass protein
2. Ligand binding usually leads to dimerization of receptors (Purves 15.6 (15.7) or Becker 14-17 (10-17).)
→ activation of the additional "recruited" proteins.
3 Dimerization activates a TK in cytoplasmic domain of receptor or in separate TK (that binds to receptor).
4. Active kinase auto-phosphorylates itself. TK adds phosphates (from ATP) to its own tyrosines -- each subunit phosphorylates the others.
5. Other proteins are activated by binding to phosphorylated TK's. (Becker 14-18 (10-18))
a. Some proteins can be activated by binding directly to TK
b. Some proteins are activated by binding indirectly -- these proteins bind to "adapter proteins" that are bound to the TK. How it happens:
(1). Adapter proteins bind directly to the phosphorylated TK
(2). Additional proteins bind to the adapters
(3). Binding to adapters
c. Proteins that bind directly to TK have certain types of domains -- usually called SH2 binding domains.
d. Recruitment. Note that the target protein to be activated comes to or is "recruited by" the TK; this is the opposite of the situation with most 2nd messengers where the messenger diffuses throughout the cytoplasm and "seeks out" the target protein to be activated. The recruitment method may be important in localizing the response to a particular part of the cell.
6. What ligands use TK receptors?
Many growth factors (such as EGF) and other paracrines, autocrines and juxtacrines act through TK receptors. Insulin, but not most other endocrines, acts through TK receptors. (See Purves 15.6 (15.7))
7. Activation of TK receptors often leads to changes in TF's and transcription. (Note this not the usual case with G-linked receptors -- their end target is usually not a TF.)
B. How do GPLR's and RTK's Compare? -- See table below for comparison of basic features of TK or TK linked receptors and G protein linked receptors. See Purves 15.6 (15.7) for structure & mechanism or Becker fig. 14-17 (10-17) for structure and Becker 14-18 (10-18) for mechanism.
|Properties of Two main types of Cell Surface Receptors|
Type of Receptor
|Property of Receptor||G protein Linked Receptor||Tyrosine Kinase or TK Linked Receptor (RTK)|
|Structure||Multipass (7X) transmembrane protein#||Single pass transmembrane protein##|
|What happens on ligand binding||Change in conformation||Usually aggregation (dimers)##|
|Receptor activates (binds to)||G protein||Self (other subunit) or separate TK|
|Nucleotide role in activation||Replace GDP with GTP||Add phosphate from ATP to tyr side chains|
|Activated TK or G protein subunit* binds to:||adenyl cyclase or
phospholipase C or
ligand gated channels
|Many dif. proteins, usually protein with SH2 domains**
Can bind a PLC → IP3 etc.
|Which 2nd messengers used?||cAMP or IP3 etc.||IP3, etc. (if any); not cAMP. Often does not use a 2nd messenger.|
|Receptors for||Epinephrine, ADH, glucagon (most endocrines)||Insulin and most GF's such as EGF (many paracrines & autocrines)|
|Usually modify||Enzymes, not TF's||Transcription Factors|
|Affect Transcription?||Not usually||Often|
|How contact target?||Broadcast||Recruitment|
# See Becker Fig. 14-4 (10-3).
## See Becker fig. 14-17 (10-17) or Purves 15.6 (15.7) & 15.3 (15.4).
*Either part, alpha or beta + gamma may be activator/inhibitor; G proteins can also be inhibitory
**SH2 = sarc homology 2 domain
Review problems 6-1 & 6-3
C. Important examples of proteins activated by binding to receptor TK's (directly or through adapters). See handout 15A.
→ IP3 → trigger cascade using IP3. This means that the IP3 pathway can be activated by both types of receptors -- GPCRs and TKs. The cAMP pathway (as far as we know) can only be activated by GPCRs.
1. PLC. Ligand binding to TK-linked receptor can activate a type of PLC
2. ras -- a famous (infamous?) protein. See Purves fig. 15.9 (15.11) or Becker 14-17 (10-18) and handout.
→ binding of adapter proteins → binding of ras to adapter proteins. Binding of ras to the final adapter protein (also called GEF = guanine-nucleotide exchange factor) catalyzes activation of ras = conversion of ras-GDP to ras-GTP.
a. What is ras? It's a small G protein introduced earlier.
(1). What makes it a G protein? It has GTP bound (active) and GDP bound (inactive) forms.
(2). What makes it "small?" It's not trimeric -- it has no subunits
(3). Ras (& other small G proteins) require other proteins to catalyze activation (binding of GTP) and to control rate of inactivation (GTP hydrolysis).
b. Why does anyone care about ras?
(1). Overactive form of ras (stuck in the activated 'on' form) can cause cancer. Implies normal ras involved in control of cell cycle. (See below.)
(2). Large % (about 30%) of tumors have overactive ras.
c. How is ras activated? Activation of receptor TK
d. What does (normal) activated ras do?
(1). Many growth factors use ras signaling to trigger the events needed for the G1 to S transition.
(2). Ras works by activating a series (cascade) of 3 protein kinases generally called MAP kinases.
(a). End result of cascade = activation of final kinase in cascade = MAP kinase or mitogen-activated protein kinase. See Purves 15.9 (15.11) or Becker 19-41 (17-39), or handout. Why bother? Achieves high degree of amplification of signal.
(b). Final kinase (MAP kinase) activates TF's → transcription of genes → mRNA → new proteins.
(3). 'Ras' is really a family of proteins whose members serve many different important functions.
e. How does overactive ras cause cancer? Allows improper G1 to S transition. Cell enters S in absence of external signal.
3. The same signal (same activated TK receptor) may trigger more than one signaling pathway. For example, EGF can trigger both the IP3 pathway and the ras pathway. (Becker fig. 14-18 (10-18)).
D. A human example of TK receptor signaling: FGF (Fibroblast growth factor) and FGF Receptor. See Becker figs.14-19 & 14-20 (10-19 & 10-20).
1. Signaling system uses a TK receptor
2. FGF & FGFR needed for proper development as described in Becker chap.10. Failure of signal transmission (or premature transmission) causes developmental abnormalities. See Becker fig. 14-20 (10-20).
3. Why is achondroplasia (type of dwarfism), due to defective FGFR, dominant? See Becker fig. 14-19 (10-19), and box on handout 15A.
a. The general principle: TK receptor monomers must dimerize in order to act. If 1/2 the receptors (1/2 the monomers) are abnormal, most of the dimers that form are abnormal.
b. An important consequence: "lack of function" mutations in TK receptors are often dominant. (For an example see Becker figs. 14-19 & 14-20 (10-19 & 10-20). Dimers form, but they are inactive. (See dominant negatives, below.)
c. This case: In a heterozygote for achondroplasia, 1/2 the FGF receptor (monomers) are defective, therefore dimers that form are defective.
(1). In the example shown in Becker & on handout, the cytoplasmic domain of the defective receptors is missing. Dimers form, but most dimers are never activated -- the two monomers can not phosphorylate each other. (Fig. 14-19) Therefore the signaling is badly disrupted. (In the example shown in Fig. 14-20, the FGF is needed to turn on formation of mesoderm, so the mutant fails to form mesoderm. This type of mutation is called a 'dominant negative' as explained below.)
(2). (FYI) In most cases of human achondroplasia, the mutation is in the transmembrane domain of FGF3 Receptor and dimers form, but act abnormally. (In these cases, the FGF signal is needed to turn on bone differentiation and turn off cell growth. Mutant dimers signal prematurely, so differentiation starts -- and cell growth stops -- before bones are long enough. This type of mutation is called a 'gain of function' mutation, because it works when it shouldn't.)
4. Dominant Negatives.
a. Definition: An abnormal allele like the one that produces the FGF receptor without a cytoplasmic domain is called a "dominant negative mutation." A dominant negative allele (or mutation) makes an inactive protein that disrupts function even in the presence of a normal allele (and normal protein).
b. 'Dominant negative' means that the heterozygote is negative for function, not that it doesn't produce any protein. There is actually a mixture of normal and abnormal protein present. What's unusual is that the abnormal, inactive, protein 'gets in the way' and interferes with the working of the normal, active, protein. So overall, there is a lack of function in the heterozygote.
c. Negative mutations (those that produce inactive protein) are usually NOT dominant. Most negative or "lack of function" alleles (or mutations) are recessive. If there is a mixture of normal and abnormal protein in the heterozygote, the normal, active, protein usually works (in spite of the presence of abnormal, inactive, protein). So usually, overall, there is NO lack of function in the heterozygote.
d. Significance: What does the existence of a dominant negative mutation imply? It indicates that the gene involved codes for a protein that must polymerize in order to act.
E. Signaling Pathways all interrelate
1. Different 2nd messengers can influence the same enzyme/pathway.
2. Each signaling system can affect the others -- For example, Ca++ levels can affect kinases/phosphatases and phosphorylations can affect Ca++ transport proteins (& therefore Ca++ levels). See an advanced text if you are interested in the details.
III. Regulation of the (Normal) Cell cycle -- see handout 15B .
A. Overview of Cell Cycle
1. Steps. G-1, S etc.; histones made in S too along with DNA.
2. Role of RTK. Usually required for transition from G-1 to S.
3. What is wrong with cancer cells? Usually G1-S switch is defective. Cycle is normal length (not shorter), but cell does not pause normally at the critical checkpoint -- proceeds too readily from G1 to S.
B. Mutants (in yeast) indicate the existence of two major control points or checkpoints. See Becker fig. 19-31 (17-30) or Purves 9.4 (9.6).
1. Near G1/S (border of G-1 & S) also called "start" in yeast or the "restriction point" in mammalian cells
a. Actual checkpoint is in G-1, near end; determines whether cell will enter G-0 (non dividing or non cycling state) or S (or pause in G-1 and await further instructions). Irreversible decision is to proceed past checkpoint and enter S.
b. This is the major checkpoint/decision point for animal cells in the adult.
c. Many growth factors (like EGF) act at this point -- signals from neighboring cells are needed to enter S.
2. G2/M (Border of G-2 & M).
a. Needed to check all components are ready for mitosis before proceeding.
b. This is the major checkpoint/decision point for cleaving eggs. (see Becker figs. 19-28 & 19-30) Cycle here is basically all S and M -- G1/S switch is in override.
c. Note: Passage of this checkpoint in animal cells generally depends on the internal state of the cells, not on presence/absence of GF's.
3. Additional checkpoints exist; see texts (especially more advanced ones) if you are interested.
C. Basic switch or regulatory protein -- similar in both cases.
1. Is regulatory protein an inhibitor or an activator? Fusions (between cells at different points in the cell cycle) imply decisions at checkpoints are controlled primarily by an "on switch" not release of an "off switch." See Becker Fig. 19-32 (17-31). You need presence of an activator signal (not loss of an inhibitor) to proceed past a checkpoint. (However, production/activation of the switch protein complex involves a complicated process which can involve multiple inhibitors and/or activators.)
2. Switch controlled by, or is, a protein kinase -- at least 2 different ones -- one for "start" and one for G2/M. In each case you need to have an active protein kinase.
3. Each kinase phosphorylates a specific set of proteins (See handout)
4. These kinases have 2 parts
→ active form. (See Becker fig. 19-35 (17-34) if you are curious about the details)
a. CDK = cyclin dependent kinase (called p34, cdc2 etc.). This is the actual catalytic protein. Level of kinase protein itself remains steady in cycling cells. (Probably degraded if cells stay in G0 = exit the cell cycle.) Inactive without cyclin; therefore catalytic activity of the kinase is dependent on cyclin (& other factors; see below).
b. Cyclin -- builds up, peaks, degraded (in proteasome), repeats (See Becker fig. 19-34 (17-33) for graph). Acts as an activator of the CDK.
c. Complex of CDK + cyclin forms; inactive; action of right combo of (additional) kinases and phosphatases
d. Different cyclins for G1/S and G2/M; usually different CDK's too (Depends on organism whether same or different).
5. Effects of kinase activation
a. Allow cells to pass checkpoint and enter next phase of cell cycle
b. Kinase phosphorylates and thus activates proteins needed to successfully complete next stage of cycle -- start DNA replication, disassemble nuclear membrane, separate chromosomes, etc. (see table on handout 15B)
6. What triggers synthesis of cyclin?
a. G1/S -- Stimulus from outside (usually a Growth Factor) plus internal state of cell.
b. G2/M -- internal state of cell -- is DNA replicated properly, etc.
D. Role of Cyclin
Switches in overall cycle as shown in texts
(Probably an over-simplified view) -- Starting at G1/S -- See handout 15B. Buildup of cyclin → Activation of CDK triggers G1→ S and then CDK is inactivated by degradation of cyclin. Process repeats at G2
1. Regulation occurs at many levels
a. Cyclin levels are regulated by controlling synthesis of mRNA (transcriptional control) and therefore protein, and by controlling degradation of both mRNA and protein (post transcriptional control)
b. Activity of many proteins involved is regulated post translationally by extensive modifications (mostly phosphorylations and dephosphorylations) -- examples include kinases and TF's.
c. Cycle is influenced by external factors (GF's, hormones, contact from other cells, etc.) and by internal factors such as state of the chromosomes, DNA damage etc. Both sets of factors alter passage through the cycle by triggering activations or inactivations of proteins (TF's, kinases, etc.). Regulation of cell cycle integrates both sets of information.
2. Why all these multiple controls and steps? If adult cell divides when it shouldn't → cancer; if fails to divide get loss of repair (no healing) and degeneration in adult. So need this very carefully controlled. So have multiple "brake" and "accelerator" proteins in this system. Many cancers traced to loss of "brake protein" or over production/activation of "accelerator protein," as discussed above; more details below.
To review regulation of the cell cycle, try Problem 15-8.
IV. Growth Factors, Growth Control, & Cancer -- Putting it All Together.
This material is covered in Becker, chap 24 ( Ch17 p. 562 to end), and Purves Chap 17 pp350-355. We'll finish it next time.
A. What is wrong with cancer cells? Usually G1-S switch is defective. Cycle is normal length (not shorter), but cell does not pause normally at checkpoint -- proceeds too readily from G1 to S.
1. Problem can be at many different steps-- with production of CDK/cyclin complex, with its activation, or with it's targets. (See handout 15B)
2. Two major types of mutations responsible -- as explained above, these result in either
a. Stuck accelerator (stimulation uncontrolled)
b. Brake failure (inhibition fails)
3. Types of mutations that cause cancer used to unravel normal G1/S signaling pathways & vice versa
B. Overview of how mutations mess up G1/S switch signaling
1. Can over-activate an activating (turn on) gene = "stuck accelerator" type of alteration -- activator is always 'on.'
a. Examples: Changes in genes for ras (so it's always in active form), or GF (always produced as autocrine), or GF receptor (always activated even without GF present), etc.
(1). Normal activator gene is called a proto-oncogene
(2). Over-active version is called an oncogene. (See Becker table 24-1 (17-2) for a list)
c. Oncogene can "over do it" 2 ways (see Becker fig. 24-15)
(1). Gene can be overexpressed -- problem is with gene expression = level of mRNA production and/or translation = level of protein synthesis. Protein is normal, but you make too much of it (or you make it at the wrong time/place). Mutation is in regulatory sequence controlling gene, not in coding part. (ex: constitutive production of a GF)
(2). Gene can be altered so protein made is constitutively active -- problem is with regulation of protein (not gene) activity -- protein is altered so it is always active. Mutation is in protein coding part of gene. Normal amount of protein is made, but protein is altered. (ex: production of altered GF receptor that is active without its ligand.)
2. Can inactive a blocking gene = "brake failure" type of alteration -- inhibitor is always 'off.'
a. Terminology; Normal "brake" genes are called tumor suppressors (see Purves 17.17 (18.17) for picture or Becker table 24-2 (17-3) for a list)
b. Examples: Most famous examples of tumor suppressors are rb and p53. (More details next time.)
(1). p53 -- protein that causes block in cell cycle in damaged cells. Normal p53 acts as 'brake' on activation of CDK/cyclin complex . (see Becker fig. 19-39 (17-40))
(2b). rb -- one of the targets of start kinase. rb protein must be phosphorylated (and inactivated) for cell to enter S. Rb 'holds up' the cycle until CDK/cyclin complex is properly activated. (See Becker fig. 19-38 (17-36)).
c. How mutation causes cancer: When p53 or rb (or other brake protein) is defective, cell keeps on growing when it should not -- in spite of damage (p53) or lack of growth factor signal (rb), etc. Result is often a tumor.
3. Types of mutations that cause cancer are used to unravel normal G1/S signaling pathways & vice versa
Try problems 15-1, 15-9 & 15-11.
C. How do Cancer Causing Mutations occur?
1. Somatic mutations can cause cancer (see Becker fig. 24-12)
a. What's a somatic mutation? A mistake in DNA replication that occurs in somatic cells, not in germ cells. Is not inherited or passed on.
b. Changes in Regulation. Genes can become over-active [or inactive] because of rearrangements or other mutations that alter effects of enhancers, silencers, etc. Example of cancer caused by rearrangements of regulatory sequences: Burkitt's lymphoma. (Proto-oncogene is placed next to a very strong enhancer.) See Becker fig. 24-13.
c. Changes in Protein Structure. Protein can become over-active [or inactive] because of changes in the gene that alter the amino acid sequence of the protein. Example caused by rearrangement: chronic myelogenous leukemia (CML -- too many white blood cells) -- cells make a hybrid, constitutively active TK instead of normal TK whose activity is regulated. See Becker Fig. 24-14 & 24-15.
2. Viruses can cause cancer -- viral DNA integrates into normal cell DNA and brings in new genes (or messes up old ones of host).
a. Virus can carry in a new gene/protein -- an oncogene or a gene that codes for the inhibitor of a tumor suppressor. See Becker fig. 24-18. (Example: How HPV causes cervical cancer.)
b. Virus DNA can destroy a host gene -- virus can integrate into DNA in middle of a normal gene and inactivate that gene -- can knock out a tumor suppressor.
c. Virus DNA can carry in a new regulatory sequence -- virus can integrate into DNA near a normal host gene and provide an enhancer or silencer that changes expression of that host gene -- can turn on a proto-oncogene.
d. Most cancers are not caused by viruses. A few types of cancer are associated with viruses (see Purves Table 17.1 (18.2)) but even in these cases the viruses alone are usually not sufficient to cause the cancer. An example: Cervical cancer is largely viral in origin, in that HPV infection is usually involved. However HPV infection is not sufficient to cause it. HPV makes a protein that inactivates rb protein. HPV is an example of case a, not b. (Note: there is some recent evidence that some cases of prostate cancer are associated with a viral infection. See also medpage for a reference to the original report.)
3. You can inherit a predisposition to cancer, not the disease itself. How can you inherit a "predisposition" = high chance of getting cancer?
a. Genes that affect DNA replication &/or repair. If you inherit versions of genes giving low repair or high mutability, tend to get cancer -- sooner or later, one of the random mutations that occurs is likely to mess up a proto-oncogene or tumor suppressor gene as above. Example: xeroderma pigmentosum, which carries a high risk of skin cancer because of defects in the genes for DNA repair. See Becker, box 24A & fig. 24A-1.
b. Tumor suppressor mutations. If you inherit one defective copy of a tumor suppressor gene, nothing happens unless the second copy of the gene gets messed up in a cell. If both copies of a tumor suppressor gene in one cell get inactivated or lost, cancer can result. (This is the "two-hit" hypothesis for how mutated tumor suppressors cause cancer. See Purves 17.16 (18.16) or Becker fig. 24-17. Example: retinoblastoma, which causes a high risk of eye and ovarian tumors, is caused by defects in rb.
4. Most cancer is sporadic, due to somatic mutation. Not inherited.
5. Cancer develops in stages
a. Most cancers have more than one mutation
b. Selection -- selection for increasing loss of growth control occurs as disease progresses -- cells that grow more aggressively (due to additional mutations) outgrow the others.
c. Progression: Normal cell → benign tumor → malignant (invasive) → metastasis (spreads). See Purves 17.18 (18.18) or Becker fig. 24-10.
d. What sort of mutation causes cancer? Is cancer caused by a 'lack of function' mutation or 'gain of function' mutation? Usually both.
Try problems 15-3 & 15-4.
Next Time: Wrap up of Growth Control, & Cancer; Then intro to endocrinology.