C2006/F2402 '06 OUTLINE OF LECTURE #11

(c) 2006 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/22/2006 11:05 AM .

Handouts:  Handouts are 11A (Big Bang Model of Signaling),  11B (Signaling with second messengers), & 11C (Cell Cycle)

Links to diagrams and animations on signaling are at http://www.columbia.edu/cu/biology/courses/c2006/links04/signallinks.html.
These links are from previous years but should still be useful.

I. Regulation at translation 

    A. How to control rate of translation? In principle:

1. Can regulate half life of mRNA (control rate of degradation). In prokaryotes most mRNA's have a short 1/2 life; in eukaryotes this is not necessarily so. Different mRNA's have very different half lives. (Note: proteasomes degrade only proteins NOT RNA's. )

2. Can regulate rate of initiation of translation (control how effectively translation starts). 

    B. Some Famous Example of Regulation of Translation.  (The principles are important; we will not go into the details.)

1. Use of regulatory protein(s) to control initiation of translation &/or half life of mRNA:  -- see notes of last time for some interesting examples.

2. Use of a regulatory RNA

a. Trans acting factors can be RNA. Not all regulatory factors are protein -- some are short RNA's. (These are usually derived from double stranded RNA -- See Becker figs. 23-35 & 23-36.)

b. How does a short RNA affect translation? Small RNA binds to mRNA Formation of double stranded RNA. This triggers degredation &/or inhibition of translation of the mRNA, and may also inhibit transcription of corresponding gene. 

c. Use in Regulation: Cells naturally produce micro-RNA's that bind to mRNA's and block translation as above. The use of short regulatory RNA's to block translation appears to be important during regulation of development. (See Becker 23-36.)

d. Use in the Lab as a tool: Called RNAi = RNA interference. The use of artificially added short double stranded (ds) RNA to block transcription/translation and turn genes off is very common. (See Becker 23-35.) Enzymes of cell convert added ds RNA into short single stranded RNA that interferes with translation and/or transcription as in b.

e. An example: See Exam #3 of C2005,  problem 1D. Question refers to effects of a mutation in the 3'UTR of a gene.  The mutation in the 3'UTR represses formation of corresponding protein, even though coding region of mRNA is normal. (I think normal levels of mRNA are made, but they aren't translated properly. Either translation of the mRNA is blocked or the mRNA is degraded.) Nothing was said about this on the exam, but it is known that the mutation affects the binding site of a micro-RNA produced during development. Probably the micro-RNA binds too well to the mutant mRNA and prevents the mRNA from being translated properly. (Most mutations in binding sites lead to weaker binding, but an occasional rare mutation leads to tighter binding.)

II. Post Translational Regulation. Don't forget: regulation occurs after translation too -- after proteins are made, they can be modified. Modifications can alter their function and/or their half life. Many examples of post translational modification have already come up and more will be discussed below. See notes of last lecture for more details.

To review post-transcriptional &/or post-translational regulation, try problem 4-13.

III. Regulation of the Cell cycle  --  see handout 11C .

Regulation of more complex event/process. We've looked at how to regulate one protein or a few at a time. Now how to regulate the cell's main event? Involves many steps, utilizing many of the different types of regulation discussed above.

    A. Review of G-1, S etc.; histones made in S too along with DNA.

    B. Mutants 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 -- 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

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 --> active form. (See Becker fig. 19-35 (17-34) if you are curious about the details)

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 above)

    D. Overall Cycle as shown in texts (Probably an over-simplified view) -- See handout 11C. Activation of CDK triggers G1 S and then CDK is inactivated by degredation of cyclin. Process repeats at G2 M (with different cyclin/CDK complex). For other pictures, see Purves fig. 9.4 (9.5) or Becker fig. 19-36 (17-35) for G2 M transition, or 19-40 (17-38) for cycle with both transitions.

1. 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 will be discussed in lecture 12 or 13.

2. Regulation occurs at many levels

a. Cyclin levels are regulated by controlling synthesis of mRNA (transcriptional control) and protein, and by controlling degradation of 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.

To review regulation of the cell cycle, try Problem 15-8.

IV. Introduction to Signaling -- How are messages sent from one cell to another? How are events in a multicellular organism coordinated?

    A. Usual Method -- one cell secretes a signal molecule that binds to a receptor inside (or on the surface of) the target cell amplification   big effect. 

    B. How do secreted signal molecules work at molecular level? Overview. See handout 11A

1. Signals are evolutionarily conserved. Same signal molecules used by different organisms for different purposes.

2. Role of Receptors. First step in signaling is binding of signal to receptor, causing conformational change in receptor.

3. Amplification = Big Bang Theory. All signals amplification = big effect from a small concentration of signal. How is amplification achieved? 

a. By affecting transcription/translation   lots of new protein   big effect

b. By cascades of modification    lots of modified protein   big effect. For examples see Purves 15.9  or 15. 15 (15.11 or 15.17. ) or Becker fig. 14-3.

c. By opening (ligand-gated) channels   a little ion flow   hit threshold voltage   open more (voltage-gated) channels   big change in ion concentrations   big effect 

4. Types of Signals. Two main kinds of chemical signals -- lipid soluble and water soluble

Signal Type Example Receptor Type Effect
Lipid Soluble Thyroxine, steroids Intracellular Gene activity
Water Soluble Peptide hormones, GF's Cell Surface Protein activity (usually)

5. Types of Receptors -- intracellular and on cell surface See Purves 15.4 (15.5)

a. Intracellular -- for lipid soluble signals. All similar, all TF's -- details below.

b. On Cell Surface -- for water soluble signals. See Becker fig. 14-2.

(1). Receptors are transmembrane proteins with an extracellular binding domain for signal. These are sometimes called "extracellular receptors" but only ligand binding domain is extracellular, not the entire protein.

(2). Three major kinds of cell surface receptors --  Listed here for reference. Details of structure/function will be discussed as we go.

(a). G Protein Linked Receptors; Also called G Protein Coupled Receptors or GPCRs. (G proteins will be discussed next time.)

(b). Tyrosine Kinase (TK) Linked Receptors. (These will be discussed later.)

(c). Ion Channels. Affects ion flow. To be discussed at length when we get to nerves. (See Purves 15.5 (15.6)).

    C. Major types of secreted Signals -- classified by type of cell that makes them and/or target location. See Handout 11B for pictures -- numbers of pictures match numbers below. (Will do/finish next time if necessary.)

1. Endocrine: Endocrine (ductless) gland secretes signal molecule (hormone) into blood target (often far away). For an example see Becker fig. 14-22.

2. Paracrine: Receptor is on adjacent cells. Act locally. See Becker fig. 14-1,  p. 414, & table 14-4 for paracrine (or autocrine) vs. endocrine. Examples:

a. histamines (mediate allergic reactions, responses to inflammation)

b. prostaglandins -- initiate uterine cramps; cause fever in response to bacterial infection.

3. Autocrine: Receptor is on same cell. ex. = some growth factors

4. Neurocrine: Neuron secretes neurotransmitter, acts on receptors on neighbor (gland, another neuron or muscle) -- like a paracrine.

5. Neuroendocrine: Neuron secretes signal molecule that acts like a hormone (travels through blood to target).

6. Exocrine: Exocrine gland secretions are released by ducts to outside of body. (Compare to endocrine.)  These secretions can carry signals --> target in different individual = pheromones (detected by olfactory receptors in mammals).

    D. Other types of Signaling

1. Gap Junctions -- allow ions & currents to flow directly from cell to cell -- used in smooth muscle synchronized contractions.

 2. Juxtacrine. Cell surface proteins from two different cells contact -- used in immune system. Similar to basic system, but signal molecule is not secreted -- remains on cell surface.

Good way to study this: Make a table summarizing C & D above. Include name of type of signaling, source of signal, type or location of target cell, any other important features, and an example of each.

Next Time: Properties of receptors for lipid soluble signals; G proteins & second messengers.