Molecular and Cell Biology Exam 3

Cell Signaling, Cell cycle, Cell death

What is DIRECT cell-to-cell signaling? Why is it important? Which proteins are involved?

• Direct contact between cells via gap junctions

• Send CHEMICAL messages

• Fast

• Free flowing

• Critical for regulating behavior of cells in animal tissues

  • Cadherins and integrins (signaling and adhesion)

• Cells express cell surface receptors to bind with signaling molecules on other cell surfaces

  • Important for embryonic development and maintenance of adult tissue

What are the three types of SECRETING cell-to-cell signaling categories? What makes the different? What are examples of each?

1. ENDOCRINE

   • Secrete hormones in the circulatory system than can act on cells at distant sites in the body

   • Example

     • Estrogen: produced in ovaries and stimulates development and maintenance of female reproductive system/characteristics

2. PARACRINE

   • Direct messaging across (not in contact) in close relation to another neighboring cell

   • Molecules released by one cell acts on neighboring target cells

   • Example:

     • Neurotransmitters

3. AUTOCRINE

   • Cell responds to signaling molecules they produced

   • Example: Immune response

Why are T-lymphocytes are considered part of the autocrine system?

T-cells respond to antigenic stimulation by synthesizing growth factor that drives its own proliferation and increases the number of responsive T-cells = amplify immune response

• abnormal autocrine signaling leads to cancer

What are steroid hormones?

• Small hydrophobic molecules

• Diffuse across the membrane

• Act by binding to receptors expressed by their target cells

  • Most receptors are expressed on cell surface

  • Some are intracellular in cytosol or nucleus

• Examples: Sex steroids, cortisol, thyroid hormone, vitamin D3, retinoic acid

What are examples of sex steroids? What is their role?

• testosterone

• estrogen

• progesterone

• *cortisol

  • secreted by the adrenal cortex

• glucocorticoids stimulate production of glucose

• all derivatives of cholesterol

What are thyroid hormones?

• synthesized from tyrosine in thyroid gland

• very hydrophobic

• important in development and metabolism

What is Vitamin D3?

• NOT a hormone

• hydrophobic

• regulates calcium metabolism and bone growth

What is retinoic acid (retinoids)?

• synthesized from vitamin A

• important for vertebrate development

What is the nuclear receptor family?

• class of transcription factors

• contained domains for ligand binding, DNA binding, and transcriptional activation

  • ligand binding regulates their function as activators or repressors of their target

• signaling molecules directly regulate gene expression

What is the pathway for when a hormone is present outside of the cell (using glucocorticoids as an example)?

1. Glucocorticoids diffuse across membrane into cytoplasm

2. Glucocorticoids bind to inactive Glucocorticoids/Hsp90 (chaperone) complex

3. Conformation change on receptor causes Hsp90 to displace

4. Receptor dimerization occurs

5. Nuclear localization signal is exposed and the dimer is transported across nuclear envelope

   • Hsp90 is blocking the NLS

6. Receptor dimer binds to recognition sites in the DNA

7. Association with coactivators with histone acetyltrasnferase (HAT) activity to stimulate tnsc

   

What does gene regulation look like for the thyroid hormone receptor?

• Thyroid hormone receptor binds DNA in either the presence or absence of a hormone

  • Presence: Hormone binding changes receptor from a repressor to an activator of target gene tnsc. The receptor associates with coactivators with histone acetyltrasnferase (HAT) activity

  • Absence: receptor associates with corepressors with histone deacetylase (HDAC) activity

What is the pathway for nitric oxide (NO) and what are its medical applications?

1. Release of neurotransmitters (ACh)

2. ACh act on endothelial cells to stimulate NO synthesis

3. NO diffuses to neighboring smooth muscle cells and activates guanylyl cyclase

4. synthesis of cGMP

5. leads to muscle cell relaxation and blood vessel dilation

medical application: Nitroglycerin used for its NO source as a means to treat people experiecing a heart attack

What are peptide hormones?

• chain of AA (few to >100)

• cannot readily diffuse across membrane

• bind to cell surface receptors

• examples:

  • epidermal growth factor (EGF; proliferation of many cell types)

  • insulin

  • neuropeptides (enkephalins and endorphins)

  • NGF (regulate development and survival of neurons)

  • PDGF (wound healing, development of embryonic tissue)

How do cells send signals across cell membranes?

ligand bind to a receptor on the cell surface which initiates intracellular rxns = signal transduction

What are G-proteins? How do they work?

• largest family of cell surface receptors = guanine nucleotide-binding proteins

• responsible for smell, sight, and taste

• General Scheme

  • binding of hormones promotes the interaction of the receptor with a G protein

  • activated G protein alpha subunit dissociates from receptor and stimulates adenylyl cyclase

  • ATP is converted to cAMP

What is the anatomy of a G protein?

• Carbohydrates: required for proper folding of receptor (stick out of the chain)

• Extracellular ligand binding domain: loop or region where specific hormones/ligand binds

• Transmembrane domains: G-proteins coupled receptors have 7 transmembrane alpha helices across cell membrane

• Intracellular ligand binding domain - Has guanine nucleotide exchange factor (GEF) activity

How are G-proteins regulated?

1. In the inactive state, alpha subunit is bound to GDP in a complex with beta and gamma

2. Hormone binding stimulates the release of GDP and its exchange for GTP

3. Activated GTP-bound alpha and beta/gamma complex dissociate from receptor and interact with targets

4. Activity of alpha is terminated by hydrolysis of bound GTP, which is stimulated by RGS proteins. Inactivated GDP-bound alpha reassociates with beta/gamma complex

How was the role of cAMP discovered?

Researchers found that the action of epinephrine was mediated by an increase in the intracellular concentration of cAMP, leading to the concept that cAMP is a second messenger

How is cAMP formed?

formed from ATP via adenylyl cyclase (stimulated by Gs) and degraded to AMP by cAMP phosphodiesterase

How is glycogen metabolism regulated by epinephrine?

1. Receptor stimulation by epinephrine leads to G protein mediated activation of adenylyl cyclase

2. cAMP activates protein kinase A, which consists of two regulatory (R) and two catalytic (C) subunits in its inactive form

3. Binding of cAMP to the regulatory subunits induces a conformational change that leads to dissociation of catalytic subunits. which are then enzymatically active

4. PKA activates phosphorylase kinase → activates glycogen phosphorylase → catalyzes breakdown of glycogen in glucose-1-phosphate

How does 1 molecule of epinephrine lead to many molecules of cAMP?

Each epinephrine molecule activates 1 receptor of Gs, but each receptor can activate up to 100 molecules of Gs which then stimulates activity of adenylyl cyclase leading to many molecules of cAMP

How does cAMP induce gene expression?

1. Receptor stimulation leads to G protein mediated activation of adenylyl cyclase, synthesis of cAMP, and activation of PKA

2. The free catalytic subunit of PKA translocates to the nucleus and phosphorylates the TF CREB (BREbinding protein), leading to the recruitment of coactivators and expression of cAMP-inducible genes

Why is regulation of gene expression by cAMP important?

plays roles in controlling proliferation, survival, and differentiation in animal cells, as well as learning and memory

How can protein kinases function in isolation in the cell?

protein phosphorylation is rapidly reversed by protein phosphatases that remove the phosphate group from tyrosine, serine, or threonine residues. Phosphatases terminate responses initiated by receptor activation, regulating cell stimulation

What are receptor tyrosine kinases?

• Enzyme-linked receptors that phosphorylate substrate proteins on tyrosine residues

• Each receptor consists of an N-terminal extracellular ligand-binding domain, a single transmembrane alpha helix, and a cytosolic C-terminal domain with TK activity

• Growth factor binding induces receptor dimerization, which results in receptor autophosphorylation as the two polypeptide chains cross-phosphorylate one another

How were receptor TK first discovered?

During a study of oncogenic protein of Rous sarcoma virus directly linking tyrosine phosphorylation to abnormal growth of cancer cells

How does signaling work for receptor tyrosine kinases?

1. Ligand induced receptor dimerization.

   • Some GF are already dimers, but others are monomers that lead to receptor dimerization due to conformational changes

2. Autophosphorylation of receptors as dimerization chain cross-phosphorylate one another. Play two key roles:

   1. Phosphorylation of tyrosine residues within catalytic domain increases PK activity

   2. Phosphorylation of tyrosine residues outside of catalytic domain creates specific binding sites for additional proteins that transmit intracellular signals downstream of activated receptors

The association of downstream signaling molecules with receptor TK is mediated by what?

Protein domains binding to specific phosphotyrosine-containing peptides

• SH2 domains

  • consist of ~100 AA

  • bind to specific short peptide sequences containing phosphotyrosine residues

What occurs when SH2-containing proteins associate with RTKs?

• localize proteins to plasma membrane

• leads to their association with other proteins

• promotes their phosphorylation

• stimulates their enzymatic activity

What are nonreceptor tyrosine kinases?

• Act by stimulating intracellular T with which they’re noncovalently associated

• Members of cytokine receptor superfamily

  • contain N terminal extracellular ligand-binding domain, single transmembrane alpha helices, and C-terminal cytosolic domain

    • HOWEVER, the cytosolic domain of these receptors are devoid of catalytic activity

    • instead, cytokine receptors function in association with NRTK, that are activated as a result of ligand binding

How does cytokine signaling work?

1. Ligand-induced receptor for dimerization and cross-phosphorylation of associated NRTK

2. Activated kinases phosphorylate receptor providing phosphotyrosine-binding sites for recruitment of downstream signaling molecules containing SH2 domains

What family are kinases associated with cytokine receptors members of?

Janus kinase (JAK) family

How does the JAK/STAT pathway work?

Key targets of JAK kinases are STAT proteins (signal transducers and activators of tnsc)

1. STAT protein are inactive in unstimulated cells and localized to cytoplasm

2. Stimulation of cytokine receptors leads to STAT protein recruitment that bind SH2 domain

3. Following association, STAT proteins are phosphorylated by JAK family kinases

4. STAT proteins dimerize and translocate to nulceus to stimulate tnsc of target genes

   • STAT transcription factors serve as direct link b/n cytokine receptors on cell surface and regulation of gene expression in the nucleus

What other family can NRTK be members of and what key roles do they play?

• Src family = Src + 8 related proteins

• Key role in signaling downstream of cytokine receptors, receptor TK, antigen receptors on B- and T-lymphocytes, and receptors involved in cell/cell or cell/matrix (integrins) interactions

  • Integrins also serve as receptors that activate intracellular signaling pathways = controlling cell movement, proliferation, cell survival, etc.

How does integrin signaling work?

1. Binding of integrins to EM leads to integrin clustering and activation of the NRTK FAK (focal adhesion kinase) by autophosphorylation

2. Src then binds to the FAK autophosphorylation site and phosphorylates FAK on additional Tyr residues, which serve as binding sites for downstream signaling molecules containing SH2 domains

Why are MAP kinase pathways important?

• major pathway of signal transduction activated downstream of both receptor and nonreceptor TK

• Cascade of highly conserved protein kinases that play a role in signal transduction in all Euk

What are MAP kinases?

• Mitogen activated protein

• activate in response to growth factors and signaling molecules

  • Yeast - control mating, cell shape, and sporulation

  • High Euk - ubiquitous regulators of cell growth and differentiation

What is the ERK family?

• Extracellular signal-regulated kinase

• Central role of ERK signaling emerged from Ras protein studies

  • Ras first identified as oncogenic proteins of tumor viruses causing sarcomas in rats

How does the Ras protein work? What does it do?

• Active Ras directly induces proliferation of normal mammalian cells; interface with Ras function blocks growth-factor-induced cell proliferation

• Guanine nucleotide-binding proteins that function analogously to G-protein alpha subunits, alternating b/n inactive GDP bound and active GTP bound forms

• Ras functions as a monomer rather than in association with beta/gamma subunit

• Ras activation mediated by guanine nucleotide exchange factors (GEFs) that stimulate release of bound GDP in exchange for GTP. Ras-GTP complex is terminated by GTP hydrolysis, stimulated by interaction of Ras-GTP with GTPase-activating proteins (GAPs)

• *In cancer, GTP hydrolysis is often inhibited by Ras proteins, leaving it continuously activated and driving proliferation

How does the Ras/Raf/ERK pathway work downstream of receptor TKs?

1. Growth factor binding. to a receptor TK leads to autophosphorylation and formation of binding sites for the SH2 domain of guanine nucleotide exchange factor (GEF)

2. This recruits the GEF to plasma membrane, where it stimulates Ras GDP/GTP exchange

3. Activated Ras/GTP complex activates the Raf protein kinase

4. Raf phosphorylates and activates MEK, a dual specificity protein kinase that activates ERK by phosphorylation on both threonine-183 and Tyr-185 residues

5. ERK phosphorylates variety of nuclear and cytoplasmic target proteins

How does ERK induce immediate-early genes?

1. Activated ERK translocates to the nucleus where it phosphorylates the TF Elk1

2. Elk1 binds to the serum response element (SRE) in a complex with serum response factor (SRF)

3. Phosphorylation stimulates the activity of Elk1 as a transcriptional activator, leading to immediate-early gene induction, in secondary response genes

Why do yeast and mammalian cells have multiple MAP pathways and what does each cascade consist of?

• controls distinct responses (ERK, JNK, p38)

• consists of 3 protein kinases: terminal MAP kinase, and 2 upstream kinases (analogous of Raf and Mek)

What helps to maintain specificity of MAP kinase signaling?

Scaffold proteins

What is PIP2?

• phosphatidylinositol 4,5-bisphosphate

• membrane phospholipid

• component of plasma membrane, localized to inner leaflet

• can be phosphorylated on the 3 position of inositol by phosphatidylinositide (PI) 3-kinase

What is the PI 3-kinase/Akt pathway?

1. PI 3-kinase is recruited to activated receptor TK via SH2 domain

2. PI 3-kinase phosphorylase the 3 position of inositol, converting PIP2 to PIP3

3. Akt is recruited to plasma membrane by binding to PIP3 via its pleckstrin homology (PH) domain

4. It is then activated as a result of phosphorylation by two other protein kinases (PDK1 and mTORC2) that also bind PIP3

5. Akt then phosphorylates a number of target proteins, including direct regulators of cell survival, several TF (FOXO), and other protein kinases, including GSK3 (which is inhibited by Akt phosphorylation)

6. GSK3 phosphorylates metabolic enzymes, the translation initiation factor eIF2B, and TFs

What is the mTOR pathway?

Regulates protein synthesis and autophagy in response to Akt signaling and the availability of energy and AA

1. Akt inhibits TSC, leading to activation of the GTP-binding protein Rheb and mTORC1 in response to growth factor stimulation

2. In contrast, AMPK activates TSC, leading to inhibition of Rheb and mTORC1 if cellular energy stores are depleted. mTORC1 is also inhibited by AA starvation

3. When active, mTORC1 stimulations tnsl by phosphorylating S6 kinase (which phosphorylates ribosomal protein S6) and eIF4E binding protein 1 (4EBP1), relieving inhibition of tnsl initiation factor eIF4E. In, addition, mTORC1 inhibits autophagy

   • eIF4E controls tnsl by binding to the 5’cap of mRNAs

   • in the absence of mTORC1 signaling, nonphosphorylated 4EBP1 binds to eIF4E and inhibits tnsl by interfering with the interaction of eIF4E with eIF4G

What is the TGF-β/Smad pathway? How is it similar to the JAK/STAT pathway?

• Similar to the JAK/STAT signaling, however the receptors for transforming growth factor beta, and related polypeptides are protein kinases phosphorylate serine/threonine residues not tyrosine

  1. Composed of two distinct polypeptides (type I and II) that become associated following ligand binding

  2. Type II receptor phosphorylates type I receptor, which in turn, phosphorylates TF of Smad family

  3. Phosphorylated Smads form complexes that translocate to nucleus and stimulate expression of target genes

What is the key role of NF-κB signaling? What do members respond to?

• Consist of five TF

• Play roles in immune system, inflammation, proliferation, and survival

• Activated in response to cytokines, growth factors, bacterial/viral infections, and DNA damage

What is the NF-κB signaling pathway?

1. In the inactive state, homo- or heterodimers of NF-κB are bound to IκB in the cytoplasm

2. Activation of the tumor necrosis factor (TNF) receptor (consisting of 3 polypeptide chains) leads to the recruitment of adaptor proteins that activate IκB kinase

3. Phosphorylation marks IκB for ubiquitylation and degradation by the proteasome, allowing NF-κB to translocate to the nucleus and active tnsc of target genes

What is the Wnt pathway in the presence and absence of Wnt?

Example of signaling system that activates TF by inhibiting their ubiquitination and degradation

Proteins are a family of secreted growth factors that bind to complex of receptors of the Frizzled and LRP families

Signaling from Frizzled and LRP stabilizes beta-catenin (transmembrane protein linking cadherin to actin at adherin junctions)

beta-catenin acts as a direct regulator of gene expression by functioning as a transcriptional activator

1. ABSENCE: β-catenin is phosphorylated by GSK3 in a complex with casein kinase1, axin, and APC (the destruction complex), leading to β-catenin ubiquitylation and degradation.

2. PRESENCE: Wnt polypeptides bind to Frizzled and LRP receptors, leading to recruitment of Dishevelled (a cytoplasmic protein), inactivation of the destruction complex, and stabilization of β-catenin. β-catenin then translocates to the nucleus and forms a complex with Tcf transcription factors, converting them from repressors to activators of their target genes.

What is the Notch pathway?

Example of direct cell/cell interactions during development

Notch is a large protein with single transmembrane domain that serves as a receptor for signaling by transmembrane proteins (delta) on surface of adjacent cells

1. Binding of delta leads to proteolytic cleavage of Notch by γ-secretase

2. Intracellular domain of Notch is translocated to the nucleus

3. Notch domain interacts with TF (CSL in mammals) and converts it from a repressor to an activator of target genes that inevitably determine cell fate

How is the activity of signaling pathways controlled?

Feedback loop

• negative feedback loop = NF-κB pathway

What is crosstalk?

Interaction of one signaling pathway with another, which integrates the activities of different pathways w/n the cell

Extensive crosstalk b/n individual signal transduction pathways means multiple pathways interact to form signaling networks within the cell

Which pathways have extensive crosstalk? How?

PI 3-kinase/Akt/mTORC1 and Ras/Raf/MEK/ERK pathways

• The Ras/Raf/MEK/ERK and PI 3-kinase/Akt/mTORC1 pathways are connected by both positive and negative crosstalk, including activation of PI 3-kinase by Ras, inhibition of Raf by Akt, inhibition of TSC by ERK, and activation of mTORC1 by ERK.

How do all cells reproduce?

By dividing into two

• each parent cell gives rise to two daughter cells → each new daughter cell can grow and divide to give rise to new cell population

What is mandatory for cell division?

It must be controlled and regulated

• Euk: cell cycle controlled by protein kinases conserved from yeasts

• High-order Euk: self regulated by growth factors

What are the 4 stages of the division cycle?

1. cell growth

2. DNA replication

3. distribution of duplicated chromosomes to daughter cells

4. cell division

How often does the cell cycle occur and what are the stages?

• Approximately divide every 24 hours

• two stages: interphase and mitosis

What is mitosis?

• Nuclear division

• Separation of daughter chromosomes, ending in cytokinesis

• only lasts about 1 hour

• 4n then 2n after cytokinesis

What is interphase? What are its phases?

• 95% of cell cycle

• Chromosomes are decondensed and distributed through nucleus

• Time at which both cell growth and DNA replication occurs

• Phases:

  • G1 (Gap 1): interval between mitosis and initiation of replication

    • 2n

    • cell is metabolically active and continuously grows

    • does NOT replicate DNA

  • S (synthesis): DNA replication

    • 4n

  • G2 (Gap 2): proteins synthesized in preparation for mitosis

    • 4n

  • *G0: division is ceased altogether or divide on occasion

    • cell as metabolically active

How does the cell cycle work for early embryos?

Cell growth does not take place, instead the cycle rapidly divides the egg cytoplasm into small cells, i.e. no G1/G2, just very short S phases alternating with M phases

How does the G1/S regulatory signal work in yeasts and animals?

Yeasts = STARTS

• once passed START, they’re committed to the S phase and undergo one division cycle

• Passage through START controlled by: availability of nutrients and cell size

• Represents a decision point at which cell determines whether sufficient nutrients are available

• START is the point at which cell growth is coordinated with DNA replication and division

Animals = restriction point

• regulated primarily through extracellular growth factors that signal proliferation

  • Presence of GF in G1 → S phase and cell cycle

  • Absence of GF in G1 → progression through cycle stops and enters G0 where they remain for long periods of time without proliferation

How does G2/M checkpoint for oocytes?

Vertebrate oocytes remain in G2 for long periods until progression to M phase is triggered by hormonal stimulation

Why are cell cycle checkpoints important? What are some examples?

• They coordinate the cell cycle

• DNA damage checkpoints make sure damaged DNA is not replicated or passed to daughter cells (occurs in G1, S, and G2)

  • G1 = allows repair of any DNA damage to take place before entering S

  • S - provide continual monitoring of DNA integrity and quality control monitor to promote repair of errors

  • G2 - prevents initiation of mitosis if the cell contains DNA that has not been completely replicated or contains unrepaired lesions

• Spindle assembly checkpoints monitor alignment of chromosomes in mitotic spindle to ensure complete set is distributed accurately to daughter cells

  • if not, the cell will arrest at metaphase until a complete set is organized

How did studying frog oocytes help understand cell cycle regulation?

• oocytes arrested in G2 until progesterone triggers entry into M phase of meiosis

• Found that G2 could be induced to enter M by microinjection of cytoplasm from hormonally stimulated oocytes

  • the cytoplasmic factor present in hormone-treated oocytes was sufficient to trigger G2/M in unstimulated oocytes = maturation promoting factor (MPF)

• MPF also present in somatic cells acting as a general regulator of transition of G2 to M

How did genetic analysis of yeast help understand cell cycle regulation?

• Identified temperature-sensitive mutants defective in cell cycle progression

• These underwent growth arrest at specific points in cycle

• cdc2 and cdc28 are homologous genes required for passage through START and entry into M and code for protein kinase. (In Euk this is known as Cdk1)

How did studying sea urchin embryos help understand cell cycle regulation?

• Following fertilization, embryos rapidly divide

• With protein synthesis inhibitors, entry into M phase required new protein synthesis

• Cyclins - function to induce mitosis, with their periodic accumulation and destruction controlling entry/exit from M phase

• *cyclin function was proven when microinjection of cyclin A into frog oocytes sufficiently triggered G2 to M

What is MPF composed of?

CDK1 and cyclin B

What is cyclin B and why is it important to the Cdk1 protein kinase?

• regulatory subunit

• required for catalytic activity of Cdk1 protein kinase

How is MPF regulated? What are the key steps?

By the phosphorylation and dephosphorylation of Cdk1

1. Cyclin B forms complex with Cdk1 during G2. As the complex forms, Cdk1 phosphorylates at two regulatory points:

   1. Threonine-161

   2. Tyr-15 (catalyzed by Wee1 which inhibits Cdk1 activity leading to accumulation of inactive complexes)

      • dephosphorylation of Tyr-15 via Cdc25 activates complexes

   3. Once Cdk1 activates, it phosphorylates target proteins initiating M phase. It can also trigger degradation of cyclin B as a result of ubiquitin-mediated proteolysis via anaphase-promoting complex/cyclosome (APC/C). Resulting destruction inactivates Cdk1 → cell exits mitosis → cytokinesis → return to interphase

What are the different families of cyclins?

G2 → M driven by Cdk1 association with mitotic B-type cyclin

START passage to S driven by Cdk1 association with G1 cyclins (Cln’s)

Cell cycles (Higher Euk) controlled by multiple cyclins and Cdk1-related Pks

• G1/S regulated by CDK-4, -6, -2, with cyclin D and E

• S/G2 regulated by Cdk2 with A-type cyclins 1 and 2

• G2/M regulated by Cdk1 complexes with A- and B- type cyclins

Cdk-1 and -2 activity inhibited by phosphorylation at Tyr-15 and activated by dephosphorylation by Cdc25. Also regulated by binding of inhibitory proteins (CKIs)

What pathways do growth factors induce the synthesis of cyclin D?

Ras/Raf/MEK/ERK

What happens to cyclin D during G1?

Begins to rapidly degrade because of APC/C ubiquitin ligase and the intracellular concentration falls

• As long as growth factors are present through G1, complexes of Cdk4, 6/cyclin D drive cells through the restriction point.

• If growth factors are removed prior to this key regulatory point, the levels of cyclin D rapidly fall and cells are unable to progress through G1 to S, instead becoming quiescent and entering G0.

How do defects in cyclin D regulation contribute to the loss of growth regulation characteristic of cancer cells?

• Rb (key substrate protein of Cdk4,6/cyclin D) frequently mutated in human tumors

• Rb is prototype of tumor supressor gene = inactivation leads to tumor development = act as brakes that slow down cell cycle progression

• Play key role in coupling cycle machinery to gene expression required for cycle progression and DNA synthesis

• Rb acts as a molecular switch that converts E2F from repressor to activator of genes required for cycle progression

How is the Rb protein regulated?

Phosphorylated: by Cdk4,6/cyclin D complex as they pass G1 restriction point

Unphosphorylated: Rb binds to members of the E2F family of TF → E2F binds to target sequences in either presence or absence of Rb → Rb acts as a repressor, so complex suppresses tnsc of E2F-regulated genes

• Therefore, phosphorylation of complex results in dissociation from E2F = activated tnsc

What regulates progress through restriction point to S phase?

Cdk2/cyclin E

• Results from cyclin E synthesis (which is stimulated after Rb phosphorylation) and activity of Cdk2/cyclin E inhibited in G0/early G1 by p27, which is relieved by multiple mechanisms:

  1. GF signaling via both Ras/Raf/MEK/ERK and PI 3-kinase/Akt pathway reduces tnsc and tnsl of p27

  2. Activated Cdk2 completes degradation of p27 by phosphorylating and targeting it for ubiquitin → further activates Cdk2/E that phosphorylates and inactivates APC/C preventing cyclin E degradation

How is the S phase regulated? What is the pathway?

Cdk2/E complex initiates S phase by activating DNA synthesis at replication origins

DNA replication initiated by MCM helicase protein activity (regulated by Cdk/cyclin complexes)

1. MCM helicase proteins bind to replication origins, with origin recognition complex (ORC) proteins during G1

2. Remain inactive pre-replication complex through G1 and becomes activated entering S phase resulting from Cdk2/E phosphorylating activating proteins recruited to pre-rep complex

3. Inhibition of APC/C (via Cdk2/E) leads to activation of DDK protein kinase that phosphorylates MCM directly

4. Activation of MCM helicase initiates replication and MCM protein moves away from origin with replication fork

5. High activity of Cdks prevents MCM proteins from reassociating with origins during S, G2 and M, so pre-rep complexes can only reform during G1 (when Cdk activity is low)

What is the pathway used by DNA damage checkpoints?

DNA damage activates checkpoint kinases, which arrest the cell cycle by inhibiting Cdc25 phosphatases

1. The ATR and ATM protein kinases are activated in complexes of proteins that recognize DNA damage. ATR is activated by ssDNA or unreplicated DNA, and ATM via ds-breaks

2. ATR and ATM phosphorylate and activate Chk1 and 2 protein kinases, respectively

3. Chk 1 and 2 phosphorylate and inhibit the Cdc25 protein phosphatases. Cdc25 phosphatase are required to activate both Cdk2 and Cdk1, so their inhibition leads to arrest at the DNA damage checkpoints in G1, S, and G2

In mammals, p53 is phosphorylated by both ATM and Chk2 which stabilize it leading to rapid increase in levels. p53 is a TF and increased levels lead to induction of Cdk inhibitor p21 that inhibits Cdk2/E,A complex

• loss of p53 function prevents cell cycle arrest in response to damage, so this passes to daughter cells

What are the states of mitosis in order?

1. Prophase

2. Prometaphase

3. Metaphase

4. Anaphase

5. Telophase (+ cytokinesis)

What defines prophase?

• Beginning marked with appearance of condensed chromosomes consisting of two sister chromatids

• These become untangles during chromatin condensation and held together at the centromere where proteins bind to form the kinetochore

• Cytoplasmic changes leading to development of mitotic spindle initiate

• Centrosomes (duplicated in interphase) separate and move to opposite sides of nucleus serving as two poles of spindle

• Nuclear envelope breakdown

What defines prometaphase?

• Microtubules attach to kinetochore of condensed chromosomes

• Kinetochore of sister chromatids oriented on opposite sides of chromosomes attaching to microtubules emanating from opposite poles of spindle

What defines metaphase?

Chromosomes shuffle back and forth until alignment on the metaphase plate at center of spindle

What defines anaphase?

Link between sister chromatids breaks and they separate to opposite poles

Cytokinesis can start in late anaphase

What defines telophase?

Nuclei reform, and chromosomes decondense

Cytokinesis almost complete at end of telophase = formation of two interphase daughter cells

How does a cell enter mitosis?

• The mitotic protein kinases Cdk1, Aurora kinase, and polo-like kinases signal the events of M phase

• Aurora and Polo kinases function with Cdk1 in a positive feedback loop

  • Cdk1 activates Aurora, which activates Polo, which in turn activates Cdk1

How does chromatin condensation occur?

Driven by condensins—members of structural maintenance of chromatin (SMC) proteins

Condensins and cohesins play a role in chromosome segregation during mitosis

1. Cohesins bind to DNA in S phase and maintain linkage to sister chromatids following replication

2. Upon entering M phase, condensins are activated via phosphorylation by Cdk1 and Aurora B kinase

3. Condensins replace cohesins along more of the chromosome so sister chromatids are only linked at centromere

4. Condensins induce condensation by forming DNA loops → formation of metaphase chromosomes

How does the nuclear envelope break down?

• Cdk1/B induces nuclear envelope breakdown by phosphorylating lamins and other nuclear membrane proteins → depolarization of nuclear lamina

• Integral membranes of the nuclear membrane are absorbed into ER and distributed to the daughter cells at mitosis

How does the Golgi apparatus break down?

Breakdown into vesicles is mediated by phosphorylation of multiple Golgi matric proteins by Cdk1 and polo-like kinases

How is the cytoskeleton reorganized?

• Results from instability of microtubules

• Beginning of prophase, centrosomes separate and move to opposite sides

  • prior to separation, they mature during which they enlarge and recruit y-tubulin and other proteins for spindle assembly

    • Centrosome maturation, separation, and spindle assembly are driven by Aurora A and Polo-like kinases

• Rapid microtubule turnover in mitosis leads to depolymerization and shrinkage resulting from phosphorylation of MT-associated proteins by Cdk1, Aurora, and polo. Interphase MT are replaced by large numbers of small MT radiating from centrosome

• Breakdown of envelope allows some spindle MT to attach to chromosomes at kinetochores to initiate prometaphase

  • proteins at kinetochore are Aurora B kinase

• MT motors direct movement of chromosomes towards minus end of spindle, drawing them closer to centrosome. Action of proteins is opposed by plus-end-directed motor proteins and the growth of spindle MT, which pushes chromosomes away from spindle poles (causes shuffling)

• MT from opposite poles eventually acting on chromosome leads to alignment on plate

How does metaphase progress to anaphase?

Activation of APC/C ubiquitin ligase

1. Unattached kinetochores lead to assembly of protein complex (mitotic checkpoint complex, MCC) that inhibits APC/C

   • MCC consists of 4 proteins: BubR1, Bub3, Mad2, and Cdc20

2. Once all chromosomes align on spindle, the inhibitory complex (activity is blocked when bound by Mad and Bub proteins in the MCC) is no longer formed and APC/C is activated by Cdc20

3. APC/C ubiquitylates cyclin B, leading to its degradation and inactivation of Cdk1. In addition, APC/C ubiquitylates securin ((an inhibitory subunit of a protease called separase), leading to activation of separase.

4. Separase degrades cohesin, breaking link b/n sister chromatids and initiating anaphase, which proceeds as a result of the action of several motor proteins associated with spindle MT

*Once cell progresses to anaphase, APC/C triggers degradation of aurora and polo, allowing cell to exit mitosis and enter interphase

How does cytokinesis occur?

• Triggered by inactivation of Cdk1 during anaphase, and activity of aurora and polo kinases

• (Yeast and animals) Mediated by contractile ring of actin and myosin II filaments that forms beneath plasma membrane

  • ring formation initiated by aurora and polo

• Contraction of actin/myosin filaments pull membrane inward, pinching off

• (Higher plant) Instead of being pinches, they divide by forming new cell walls and membranes inside cell (cell plate)

What is apoptosis?

Programmed cell death

What characterizes early development?

Rapid proliferation of embryonic cells that differentiate into specialized cells of adult tissues and organs

How do differentiation and proliferation compare?

Increased rate of differentiation = decreased rate of proliferation

What phase do are most cells arrested in?

G0

How is proliferation able to occur in cells that have differentiated?

Most differentiated cells no longer have the ability to proliferate, but some retain the ability to repair damaged tissues through organism’s life. Examples:

• fibroblasts, endothelial cells lining blood vessels, smooth muscle and contractile portions of organs, epithelial cells of some internal organs

  • Endothelial cells are stimulated to proliferate by vascular endothelial growth factor (VEGF). VEGF is secreted by cells deprived of oxygen, leading to outgrowth of new capillaries into tissues lacking an adequate blood supply

  • Liver cells are normally arrested in G0 but resume proliferation to replace damaged tissue. If 2/3 of a rat’s liver is removed, the remaining cells proliferate to regenerate the entire liver in a few days

How do stem cells divide?

Stem cells divide to produce one daughter cell that remains a stem cell and a second that proliferates and differentiates

• typically five rise to rapidly proliferating cells (transit-amplifying) that undergo several divisions before differentiating

What is characteristic of stem cells and their roles in the body?

Self renewing

Role is evident in blood cells, sperm, epithelial cells of skin, and lining of digestive tract which all have short lifespans

Is it accurate to say fully differentiated cells proliferate?

No, fully differentiated cells do not themselves proliferate; instead, they are continually renewed by proliferation of stem cells

How were stem cells discovered?

Identified in the hematopoietic (blood forming) system in experiments showing that single cells derived from mouse bone marrow could proliferate and give rise to multiple differentiated types of blood cells