Note: in spring 2010, we begin exam 3 translation mechanisms (see review sheet two). Also, we did not go into as much detail about all aspects of the cell cycle as reviewed here. Consult your notes. They are what is required for you to know about that process.

I.      Control at the level of mRNA stability

        A.    The 3' untranslated region of eukaryotic mRNAs can contain repeated AU sequences
               that signal the poly-A tail to be rapidly degraded by nucleases.
            1.    This causes these mRNAs to have very short half-lives
            2.    The proteins coded for by these mRNAs are usually central to growth regulation for
                    the cell.

        B.    There also is degredation that begins at the poly-A tail and degredation that begins at the 5' region when the cap is removed.
 
        C.    Example we looked at was the mRNA for the transferin receptor.
            1.    Has several AU repeats in its 3'UTR.
            2.    Allows the formation of stem-loops that contain an iron-response element (IRE) that bind aconitase.
            3.    An IRE binding protein, aconitase, exists in two possible conformations.
                   -when iron amounts are low in the cell, it is active and binds to the IREs.
                   -This protects the mRNA from sigalling for degredation.
                   -Therefore, translation will occur and more transferin receptor will be made.
                   -This will allow more iron to be brought into the cell.
                    -When iron levels are high, no more iron entry is needed. It does not bind and the mRNA
                        degrades.
 

  II.    Signal Transduction

    A.    Three overall types
        1.    Endocrine: hormone made far away and brought through blood to target cell.
        2.    Paracrine (Contact-dependent):  Cells are adjacent. Signal secreted from cell or part of its membrane. Then
                interacts with target cell next to it.
        3.    Autocrine: Cell that makes the signal has receptors itself for that signal.

    B.    G stimulatory system: Receptors are 7-pass transmembrane proteins.
        1.    Receptor changes shape upon activation by signal.
        2.    Binds to G stimulatory.
        3.    Causes alpha subunit (bound to GDP) to disengage form beta-gamma subunit.
        4.    Causes GDP to leave and it is replaced by GTP.
        5.    Allows alpha subunit to bind and activate adenylate cyclase.
        6.    Adenylyl cyclase catalyzes formation of cyclic AMP from ATP.
        7.    When hormone no longer bound to receptor, alpha subunit hydrolyzes GTP to GDP
                causing reversal of the above events and a return to the beginning.
        8.    Cyclic AMP binds cyclic AMP-dependent protein kinase.
        9.    Causes regulatory subunits to displace from catalytic subunits, activating them.
        10.    Phosphorylation of substrates alters their activities. For example, glycogen synthase is inhibited to prevent glycogen
                from being formed from glucose and glycogen phosphorylase kinase is activated which then
                 phosphorylates glycogen phosphorylase to activate it resulting in the release of glucose from glycogen.
        11.    G inhibitory is similar to G stimulatory with the difference being that the alpha
                subunit inhibits adenylate cyclase activity thus lowering cyclic AMP levels.

    C.    Gq system (phosphatadyl inositol system)
        1.    Receptor binds signal and activates phospholipase C.
        2.    Phospholipase C beta cleaves PIP2 in the membrane.
        3.    Produces Diacylglycerol (DAG) which remains in the membrane and activates
                protein kinase C.
        4.    Also produces IP3 which is released into the cytoplasm and binds to calcium channels
                in the ER membrane, causing the release of calcium into the cytoplasm from the lumen.
        5.    Loss of calcium from the ER sends a signal to a calcium channel in the cell membrane, causing it to allow
                more calcium to enter the cell, augmenting the response.
        6.     Calcium binds to calmodulin. This complex binds to other proteins and alters their
                activity, transducing the signal. Example is CAM Kinase II which gets partially active this way and then can phosphorylate itself to become fully active. It                  can the remain 80% active even after the calmodulin complex leaves, until the activating phosphate is removed. May be involved in memory/learning.
         7.   Calcium binds protein kinase C in the cytoplasm and causes it to move to the membrane where DAG can activate it.

    D.    Receptor tyrosine kinase system  (Note: a receptor tyrosine kinase can also activate the phosphatadyl inositol system described above..)
        1.    Receptor tyrosine kinases are transmembrane proteins that are single proteins until
                activated by a signalling ligand.
        2.    When bound, they dimerize with identical receptor tyrosine kinase.
        3.    They autophosphorylate their cytoplasmic catalytic domains at tyrosines (each on the
                other).
        4.    Proteins with SH2 domains bind to the phosphotyrosines.
        5.    Proteins with SH3 domains (rich in prolines) bind these or, alternatively, the SH2 domain protein may
                itself have an SH3 domain.
        6.    SH3 domains bind other proteins activating them and transducing the signal.
        7.    Ras is one protein that is so activated.
        8.    Ras bound to GDP is inactive. When a GEF protein binds it the GDP is displaced.
        9.    GTP binds and ras can then bind other proteins and activate them.
        10.    Ras binds to GAP which accelerates ras's GTPase activity and GTP is hydrolyzed.

    E.    Drosophila R8 photoreceptor cell activation by the R7 cell
        1.    BOSS on the membrane of the R7 cell  binds its receptor sev, a receptor tyrosine kinase..
        2.    The receptor dimerizes and autophosphorylates.
        3.    Drk binds the receptor through its SH2 domain (has both SH2 and SH3 domains).
        4.    Sos binds to the SH3 domain and is activated.
        5.    A GEF (sos) binds ras causing it to displace GDP, thus actiavating ras (see above).
        6.    Ras activates Map Kinase Kinase Kinase (raf) by bringing it to the membrane area and binding to it, causing it to partially release from 14-3-3 and
                become active as a kinase.
        7.    Raf (a serine-threonine kinase) phosphorylates Map Kinase Kinase (mek), activating it.
        8.    mek (a serine-threonine and a tyrosine kinase) phosphorylates MAP Kinase (erk).
        9.    Erk dimerizes and activates p-90 (elk) which  enters the nucleus and activates SRF transcription factor. Erk dimers also enter the nucleus and there
                activate TCF. 2 SRFs and 1 TCF can then interact with the SRE enhancer element and activate transcription of early response genes such as c-myc.

III.    Cell-Cycle

   A.    Prior to cell division signals cause transcription of early-response genes like myc by phosphorylating specific
            transcription factors that then activate transciption of delayed response genes by also phosphorylating their
            specific transcription factors.

    B.    Cells move through G1, S, and G2 (together these are called interphase)

    C.    Go is what we call the state where a cell is not preparing to go into cell division, but is alive and well.

    D.    Cyclin/cdk complexes target other proteins for phosphorylation and that causes the cell-cycle to move forward
             through the restriction point in G1 and beyond.
            a.    restriction point stops the cell division from moving forward. Once past it, the decision is made unless
                   damage or other problems activate checkpoints (see below).
             b.  cdks are activated by binding a cyclin and by an activating phosphorylation at threonine 160. They are inhibited by inhibitory
                   phosphorylations of threonine 14 and tyrosine 15  and by association with cdk inhibitors. Both activation and inhibition are also controlled by
                proteases that remove target phosphates.

    E.    In mid G1 they activate S-phase cyclins and cdks. Held in check by inhibitor.

            a.    Also begin to phosphorylate Rb.
    F.    Late G1 they induce degredation of S-phase inhibitors.
            a.    phosphorylate S phase inhibitor, relieving its inhibition of S phase cyclin cdks. Targeted for degredation
                    by the ubiquitin ligase, SCF.
            b.    they further phosphorylate Rb causing it to unbind E2F transcription factor which then activates transcription of
                    itself and of more cyclin cdks as well as genes involved in DNA synthesis and the S phase cyclin which feeds back to increase Rb phosphorylation to                     completely inactivate it.

    G.    S-phase cyclin cdks phosphorylate regulatory sites in DNA pre-replication complexes on oris (assembled during
            G1.
            a.    stimulates DNA replication
            b.    prevents new pre-replication complexes
            c.    guarantees only 1 DNA replication
             d.    details:  In G1 the orc is at the ori in a non-phosphorylated form that allows binding of cdc6 and Cdt1. These recruit mcm helicase complex.                                     Forms the prereplicative complex.Then Scdk and another kinase help the pre-initiation complex to form. It also causes targeting of cdc6 for                                  degredation. Geminin binds Cdt1 and inhibits it. The Scdk also phosphorylates the orc and helps the preinitiation complex to form and join. All other                         elements needed for  DNA replication join and it ensues. Since the phosphorylated orc cannot rebind new cdc6 or Cdt1, only one replication can be                     initiated at each origin. Later, during late mitosis and early G1, the phosphates are removed, more cdc2 is made and geminin levels fall so Cdt1 is                         available. New pre-replicative complexes can then form.

    H.    Mitotic cyclin cdks made in S and G2 
            a.    held in check by phosphorylation until DNA synthesis is done.
            b.    activated by dephosphorylation
            c.    phosphorylate proteins involved in prophase and metaphase

    I.    Anaphase Promoting Factor (APC) moves cells from metaphase into anaphase and then degrades the mitotic cyclins.
            a.    a ubiquitin ligase targeting securin for degredation. Specificity factor is cdc20.
            b.    securin prevents splitting of the centrosomes to initiate anaphase and movement of sister-chromatids
                    to opposite poles. Does this by binding separase, preventing it from disrupting the cohesin complex holding the sister chromatids together.
            c.    also causes degredation of the mitotic cyclins. Specificity factor in that stage is cdh1.
            d.    Some phosphatases then remove phosphates from multiple proteins, returning the cell to G1 (or Go).

    J.    Early in next G1, phosphatases remove phosphates from pre-replication complexes which can then reassemble
            at ori regions.

    K.    Early in G1, p27, an inhibitor, is bound to cdk2/cyclin E. It leaves and joins another complex which then phosphorylates it and
             it is degraded. cyclinE/cdk2 then activates mcm helicase and causes initiation of replication at oris.

    L.    Important checkpoint proteins operate at critical junctions in the cell cycle to stop it when necessary.
            a.    DNA damage checkpoints found at mid-G1; late G1; late S phase, and late G2.
                    - all begun with ATM or ATR protein kinase phosphorylating p53 through Chk1/2..
                    - prevents mdm-2 from targeting p53 for degredation.
                    -p53 stabilized and activates the p21-CIP gene.
                    -p21 binds to most cyclin cdks and inhibits them.
                    - in late G1, late S, and late G2, another pathway is activated also by ATM or ATR.
                    - It phosphorylates Chk1 or 2 protein kinases. These phosphorylate ckc25A or C.
                    -This inhibits cdc25A or C phosphatase.
                    -This stops the removal of critical phosphates in cyclin cdks that activate their activity.
                   -Chk1 also phosphorylates p53 to further interfere with its binding to mdm-2.
          b.   Excessive myc signalling can activate the arf protein which then binds to mdm2 causing it to release p53. p53 is thus stabilized and can initiate cell-cycle                     arrest as described above or, alternatively, can help initiate apoptosis.

            b.    Unreplicated DNA checkpoint works at late G2.
                    - ATR kinase activity activated when associated with replication forks.
                    - phosphorylates Chk1 kinase
                    -Chk1 kinase inactivates cdc25C phosphatase
                    - this inhibits the cyclin cdks that activate mitosis.

            c.    spindle-assembly checkpoint prevents premature entry into anaphase
                    -Mad2 associates with kinetochores
                    -binds cdc20, inhibiting its activity in activating APC and securin ubiquitination.
            d.    chromosome segregation checkpoint
                  -Tem 1 G protein associates with centrosomes.
                    -when segregated daughter chromosomes at end of anaphase are correctly located it gets bound to GTP.
                    -this triggers a kinase cascade and activation of a cdc that causes the cell to exit mitosis.
                    -if segregation of chromosomes is not correctly completed, Tem 1 remains inactive in its GDP form.

IV.    Apoptosis
    A.    2 types of cell-death.
        1.    Necrosis: due to trauma.
            a.    cells lyse and die
        2.    Apoptosis
            a.    programmed cell-death
            b.    cells commit suicide

    B.    Survival Factors
        1.    Survival signals (trophic factors) that must be present to prevent apoptosis in cell culture systems.

    C.    Apoptosis Mechanism
        1.    Identified proapoptotic factors and anti-apoptotic factors.
             a.   anti-apoptic factors like bcl-2
             b.   BH3 domain only pro-apoptotic factors
             c.   multi-domain pro-apoptotic factors (123 proteins)
       2.   BH3-only initiates apoptosis by binding to anti-apoptotic factor, thus removing it from binding the multi-domain
             pro-apoptotic factor which can then cause apoptosis.

    D.    Effectors of the apoptosis pathway
        1.    Caspases
            a.    cysteine proteases that cleave to the COOH side of aspartic acid residues in target
                    proteins.
            b.    can be activated extrinsically as described below.
            c.    can be activated intrinsically as described below. Procaspases are cleaved to active caspases by an initiator caspase.

    E.    Cytochrome c involvement (intrinsic pathway)
        1.   Usually located between the inner and outer mitochondrial membranes.
        2    Released to cytoplasm during apoptosis most likely through pores formed by multimers of the proapoptotic multidomain factors.
        3.   Binds Apaf-1 initiating formation of the apoptosome. This involves formation of a multimer facilitated by an ATP/ADP/ATP cycle and by interacting                     CARD domains on Apaf1.
        4.   Initiates a caspase cascade by recruiting procaspase 9 molecules to the apoptosome through their own CARD domains, causing them to cleave each other                 to form active initiator caspase 9s.  These then cleave other procaspases, activating them as executioner caspases which then degrade multiple proteins in                 the cell leading to cell death.

    F.    Inhibitors of the IAFs also get released from the inner membrane mitochondrial space and counteract the inhibition of apoptosis
            caused by the IAFs (bind to aberrantly activated caspases to inactivate them).

    G.    Survival Factors induce inactivation of the pro-apoptotic regulator, Bad, a BH3 only pro-apoptotic factor. (Anti-apoptotic pathway)
        1.    Induces its phosphorylation
                a.    receptor tyrosine kinase binds survival factor and activates PI3 kinase which binds Akt kinase an PDK1 through PH domains.
                b. PDK1 and another kinase mTOR phosphorylate and activate Akt.                                                                                                                                              c. Akt dissociates in active form and phosphorylates Bad.
        2.    Phosphorylated Bad binds to 14-3-3  in the cytoplasm.
        3.    Therefore it cannot bind to the Bcl2 or Bclx  in the mitochondrial membrane.
        4.    If Bad does not bind, the Bcl2 or Bclx can inhibit the Bax/Bax or Bax/Bak formation of the cytochrome c
                channel.
        5.    This prevents release of cytochrome c and the apoptotic events outlined below.
        6.   Transcriptional inducer FOXO is also regulated by 14-3-3 binding as described above for Bad. Prevents it from
                inducing transcription of other pro-apoptotic genes.  (we did not discuss this)
       7.   Akt activation also helps activate the mTOR/raptor pathway.
             a.   inhibits TSc2/TSc1. Thus it cannot inhibit Rheb.
             b.   Rheb then activates mTOR which activates S6 kinase.
             c.   S6 kinase activate S6 ribosomal protein, activating translation.

    H.    The intrinsic apoptotic pathway
        1.    Bad (domain 3 only protein) is bound to bcl2 or bclxl.(anti-apoptotic proteins).
        2.    They then cannot inhibit the Bax/Bax or Bax/Bak (123 proteins)from forming  multimers with the cytochorome c channel.
        3.    It forms a cytochrome channel.
        4.    cytochrome c is released into the cytoplasm
        5.    cytochrome c binds to Apaf1 and caspase 9 is activated, as explained above.
       6.   can also have this intrinsic pathway activated through signals from the TNF receptor (see below). (Not covered this semester)
             a.   adaptor-bound caspase 8 activates bid (domain 3 only protein),
             b.   bid binds bcl-2, allowing the bax/bak channel to form. (see above). 

    I.   The extrinsic apoptotic pathway 
       1.   Cell-death inducing factor, for example, TNF or the Fas death receptor on killer lymphocytes binds receptor.
       2.   Receptor trimerizes, forming binding sites for and adaptor protein. For example, the death domains on the Fas death receptor interact with the same                         domains on the FADD adaptor protein. The death effector domains on the adaptor then bind to death effector domains on procaspase 8 or 10.
       3. This causes cleavage of procaspase 8 or 10 and formation of active caspases 8 or 10. These initite caspase activation of other executiner caspases and lead             to apoptosis.

    J.   p53 promotes apoptosis.
       a.   In response to DNA damage, ATM kinase phosphorylates chk2 kinase which phosphorylates p53 and stabilizes it.
       b.   p53 activates transcription of two of the BH3 only pro-apoptotic protein genes.  (Puma and Noxa)

    K.   The c-elegans apoptosis pathway. (We did not cover this in 2010.)

          a.   EGL-1 binds to anti-apoptotic CED-9
          b.   CED-9 can no longer bind and inactivate the CED-4 dimer.
          c.   CED-4 forms a tetramer.
          d.   CED-4 tetramer activates CED3 zymogen (inactive protease) and it becomes active CED-3
          e.   active CED-3 cleaves targets and causes apoptosis.

V.    Historic experiment identifying human cancer genes. (we did not discuss this in 2010)
        1.    DNA removed from human tumors.
        2.    Used to transfect normal mouse cell monolayers.
        3.    Foci resulting picked and DNA extracted again.
        4.    Used to transfect new mouse cell monolayer.
        5.    Resulting foci picked and used in shotgun cloning into bacteriophase vector library.
        6.    Phage library used to infect bacterial cells.
        7.     Resulting plaques transfered to filters and tested for presence of human Alu sequences.
        8.     Positive plaques identified and used for further DNA sequencing, etc.
        9.     Identified mutant ras first. Other human oncogenes similarly identified.
       10.   co-transfection of ras plus myc and subsequent injection of foci into mice showed a dramatic synergistic effect of having both oncogenes present in
                the formation of the tumors.

VI.    Cancer

    A.    Oncogenes
        1.    Formed from proto-oncogenes
            a.    Normal cellular proteins that have become deregulated in some way.
            b.    Are dominant, gain-of-function mutations or changes.
        2.    Originally studied in retroviruses that were oncogenic.
            a.    viral src protein has a point mutation that does not allow it to be inactivated.
            c.    viral src was transduced from a host cell during retroviral replication.
            d.    Retroviruses are RNA viruses that go through a ds DNA intermediate that integrates
                    into the host cell's chromosomes.
             e.    Another example is the viral abl gene that was transduced into Maloney mouse leukemia virus
                     resulting in unrestricted kinase activity (we did not review this in 2010)
         3.    Oncogenes can form also by gene amplicifation.
            a.    detected by the presence of double-minutes or hsr (homogeneously staining region on chromosome
                    spreads, indicating  multiple copies of the gene. Examples: mdm-2, c-myc
          4.    Oncogenes can form by chromosomal translocations
             a.    Example: translocation between chromosomes 8 (myc) and 14
                        (antibody genes). The powerful antibody gene enhancers abnormally activate
                        the translocated myc.
              b.    Example: translocation between 9 and 22, forming a bcr-abl fusion, causing continuous kinase activity. Forms the Philadelphia chromosome.
                     Imantibid (Gleevec) is a drug that can bind the active site of the bcr-able kinase and inactivate it.                              
         5.    Oncogenes can form by insertional mutagenesis
                a.    retrovirus integrates near a proto-oncogene. Its powerful enhancer activates the proto-onc into an onc.
        6.Oncogenes can also form by spontaneous mutations of all types. (Examples include the neu oncoprotein and the erbB oncoprotein
             These are mutated forms of the Her-2 and eGF receptors by either point mutation (Her-2 val-glu that causes uncontrolled dimerization) or
             deletion (EGF receptor that loses its signal binding domain and is always activated). (We did not review these particular oncogenes in 2010)

    B.    Tumor-Suppressor Proteins
        1.    Are recessive.
        2.    Must lose both copies to have an effect.
        3.    Involved in inhibiting growth signals, inhibiting progression of the cell-cycle, in DNA repair,
               in inducing apoptosis, and in causing cell-cycle arrest.
        4.    Some examples
            a.    A mutated GAP that cannot help ras hydrolyze GTP. The GAP therefore is a
                    tumor-suppressor protein. Also, mutations in ras itself.
        5.    Some examples in regulating the cell-cycle or apoptosis.
            a.    p16, p27, and p21 all bind to cyclin/cdk complexes and thereby prevent them from
                   phosphorylating their target proteins.
            b.    One target is the RB (retinoblastoma) protein which when not-phosphorylated binds to
                   a transcription factor family called E2F and prevents the activation of genes involved
                   in further cell-cycle progression, usally from G1 into S.
                    -Cdk/cyclins phosphorylate Rb. These cyclins are activated in response to mitogenic stimuli.
            c.    RB itself is a tumor-suppressor protein since it prevents cell-cycle progression when
                   bound to E2F.
            d.    p53 is a tumor-suppressor protein involved in multiple ways.
                -when induced by events that cause damage to DNA, e.g. irradiation, it can cause the
                cell-cycle to arrest by inducing the transcription of p21, a cyclin-cdk inhibitor.
                   -It also can induce apoptosis in cells alternatively, especially if the DNA damage is
                   extensive. It activates several BH3 only pro-apoptotic genes (Puma, Noxa).
                  -It therefore helps maintain the integrity of the genome of the cell, preventing the
                   accumulation of genetic damage that would occur if cells were allowed to divide in the
                   presence of such damage.
        6.    Some are proteins that are directly involved in DNA repair mechanisms. (Some texts calls them caretaker genes and considers them a separate
                category of tumor-suppressor-related genes.)
                a.    nucleotide-excision repair proteins and mismatch-repair proteins have
                       been identified as lost in many cancers. Also homologous replication-dependent repair proteins (BRACA-1 and 2).
        7.    Some are  proteins that are in the pathway that leads to apoptosis. This can
               also include p53 (see above).
        8.    Several DNA Tumor Viruses bind to these tumor-suppressor proteins to inactivate them
               when the virus transforms cells. 
                a.    Example:    SV40 which binds both to p53 and to Rb through its large T antigen.
                b.    Example:    Adenovirus which binds p53 through its E1b antigen and Rb through its
                                        E1a antigen.
                c.    Example:    Human Papilloma Virus (HPV) which binds to Rb through its E7 antigen
                                        and binds to and causes the degredation of p53 through its E6 antigen.

    C.    Other Cancer-related events in the cell
        1.    Angiogenesis contributes to tumor-growth  (We did not talk about this in our class this year).
            a.    This formation of new blood vessels to feed the tumor is promoted by factors secreted
                    from the tumor. Example: VEGF
            b.    Anti-angiogenic factors show promise of inhibiting this blood-vessel formation, thus
                   starving the tumor. example: Avastatin and Endostatin.
            c.    Primary tumors secrete inhibitors of angiogenesis around secondary tumor sites! (Not covered in 2010)
        2.    Cancer is a multi-step process.
            a.    Roughly 5-6 genetic changes in growth-control pathways must occur to form a highly
                   aggressive cancer in humans.
            b.    Both oncogene activation and tumor-suppressor loss must occur.
            c.    The latest stages involve metastasis-related mechanisms that are poorly understood but
                   allow transformed cells to break free from their tissues and move to other sites.
        3.    The drug Gleevec (ST1-571) (Imantibid) is a promising anti-cancer drug.
            a.    Chromosomal translocation between 22 and 9 cause the bcr-abl fusion protein.
            b.    Causing continuous tyrosine kinase activity.
            c.    Leads to chronic myelogenous leukemia in children
            d.    Gleevec binds to the bcr-able protein, preventing it from binding to its substrates.
            e.    This stops the signal pathway and the run away cell division it leads to.
            f.    Gleevec seems to interact with other such kinases and may help treat other cancers that involve
                    this type of over activation.
         4.    Other drug therapies include retinoic acid against acute promyelocytic leukemia caused by another (not covered in our class)
                translocation that fuses the retinoic acid receptor to a gene called PML; herceptin, a monoclonal antibody that interferes
                with the erbB-2 oncogene product (a protein kinase); and Erbitux, a monoclonal antibody that interferes with the Epidermal
                Growth Factor receptor (erbB).  Also getifinib which  is a small molecule inhibitor of the Epidermal Growth Factor receptor. (Not covered in 2010)