Lecture 3: Cellular Communication and Signal Transduction Part 2

True or False: Stimulation of adenylyl cyclase leads to an 5-fold intracellular increase in cAMP within 5 seconds.

true

What do the effects of increased intracellular cAMP depend on?

• the subcellular location of increased intracellular cAMP

• the function of the specific cell

What does protein kinase A (PKA) do?

catalyzes the transfer of a phosphate of ATP to specific serine/threonine residues on substrate proteins (phosphorylation)

What does PKA phosphorylation do?

• influences localization

• activity of protein

How is PKA activity regulated?

regulatory subunits constitutively inhibit PKA

What happens when cAMP binds to regulatory subunits?

• a conformational change and decreased affinity for catalytic subunits

• catalytic subunits are now free to phosphorylate proteins

cAMP-dependent kinase (PKA) is composed of which two subunits?

• regulatory (R)

• 2 catalytic (C)

True or False: Binding of cAMP to the regulatory subunits induces a conformational change that reduces their affinity for the catalytic subunits.

true

Where can the catalytic subunits of PKA enter?

the nucleus

What does CRE sites stand for?

cAMP response elements sites

What binds CRE sites?

CREB

What phosphorylates CREB?

PKA

What results when PKA phosphorylates CREB?

• an increase in affinity of CREB for coactivator CBP

• 10 to 20-fold increase in CREB’s ability to induce transcription of a gene with a CRE site

What does CBP stand for?

CREB-binding protein

What terminates the transcriptional signal? What happens?

phosphoprotein phosphatase 1 (PP1); PP1 dephosphorylates CREB

Where are phosphatidylinositols (PIs) mostly located?

the internal leaflet of the membrane

Inositols can be phosphorylated to yield which two major phosphoinositides?

• PIP2

• PIP3

True or False: DAG stays in the plane of the membrane, and PKC travels to the membrane to bind to DAG and get activated.

true

Water-soluble IP3 travels to the ER and does what?

stimulates Ca2+ release

True or False: Phosphatidylcholines (PCs) are abundant in the cell membrane and are DAG sources.

true

How is DAG produced from PC (2 ways)?

• direct: PLC can convert PC to phosphocholine and DAG

• indirect: phospholipase D (PLD) can convert PC to choline and phosphatidic acid (PA) → PA is converted to DAG by PA-phosphohydrolase

True or False: IP3 travels through the cytosol and binds to the IP3 receptor (IPTR), a ligand-gated channel on the ER membrane.

true

What can the rises in intracellular calcium be?

• brief or persistent

• oscillate repetitively

• spread across groups of coupled cells

Intracellular calcium remains high until what?

until IP3 is dephosphorylated and IPTR closes

Increased intracellular Ca2+ stimulates activation of what?

SERCA (sarcoplasmic and endoplasmic reticulum calcium ATPases)

What does SERCA do?

pumps Ca2+ back into the ER

What mediates the effects of changes in intracellular calcium?

Ca2+-binding proteins such as calmodulin (CaM)

What does CaM bind?

4 Ca2+ ions

What does a conformational change in CaM allow?

binding to other proteins

True or False: CaM forms a complex with many eznymes and confers Ca2+-dependence on their activity.

true

What do CaM kinases (CaMKs) do?

phosphorylate serine/threonine residues on a variety of proteins

Protein kinase C (PKC) has 10 members and is divided into which 3 groups?

• classical: require both DAG and Ca2+ for activation

• novel: require DAG only

• atypical: independent of both DAG and Ca2+

What do the effects of PKC activation depend on?

• the isoform of the enzyme

• levels of Ca2+

• DAG

Which type of PKC is PKC-alpha?

classical PKC

Describe PKC-alpha in its inactive form.

soluble and cytosolic

True or False: DAG and Ca2+ bind to the PKC regulatory domain.

true

What happens after PKC-alpha translocates to the membrane?

1. PKC-alpha kinase domain gets activated

2. PKC activation sustained beyond initial Ca2+ elevation (PLD-mediated production of DAG)

3. drives responses including proliferation and differentiation

How do phorbol esters promote tumors?

via PKC

What type of products are phorbol esters?

plant products

True or False: All phorbol esters share the ability to bind to the regulatory domain and activate PKC.

true

What do phorbol esters mimic?

mimics the DAG stimulation of PKCs

True or False: Action of phorbol esters is sustained → prolonged activation of PKCs.

true

What releases arachidonic acid (AA) from glycerol-based phospholipids?

phospholipase A2

What is arachidonic acid (AA) converted into?

eicosanoids (biologically active metabolites)

What are some examples of AAs?

prostaglandins and leukotrienes

What are the five major classes of cytoplasmic catalytic receptors?

• receptor guanylyl cyclases

• receptor serine/threonine kinases

• receptor tyrosine kinases (RTKs)

• tyrosine-kinase-associated receptors

• receptor tyrosine phosphatases

What does guanylyl cyclase do?

converts GTP into cGMP and PPi

What is an example of a receptor guanylyl cyclase?

atrial natriuretic peptide (ANP) receptor

Describe the two domains of receptor guanylyl cyclases.

• extracellular binding domain: single membrane-spanning segment

• intracellular domain: 2 consensus catalytic domains for guanylyl cyclase activity

True or False: ANP binding → conformational change in receptor → receptor dimerization and activation.

true

What does a rise cGMP levels activate?

cGMP-dependent kinase (PKG)

What does serine/threonine kinase do?

converts Ser/Thr and ATP to phosphorylated Ser/Thr and ADP

What is an example of a serine/threonine kinase?

transforming growth factor-beta (TGF-beta) receptor

True or False: Receptor Ser/Thr kinases are a part of a superfamily of glycoproteins that control cell growth and differentiation.

true

True or False: The ligand binds to Type II receptor → formation of complex of ligand, Type II receptor, and Type I receptor.

true

What does Type II receptor phosphorylation activate?

Ser/Thr kinase activity of Type I receptor

What does tyrosine kinase do?

Tyr and ATP is converted to phosphorylated Tyr and ADP

True or False: NGF binding → dimerization → direct association and trans-phosphorylation of adjacent kinase domains.

true

True or False: Activated receptors catalyze addition of a phosphate to Tyr residues on the receptor itself plus the membrane-associated and cytoplasmic proteins (downstream effectors).

true

What does tyrosine kinase-associated receptors do?

Tyr and ATP is converted to phosphorylated Tyr and ADP

How are TKARs different from RTKs?

TKARs don’t have intrinsic kinase activity (the receptor itself doesn’t contain a kinase domain)

True or False: Tyrosine kinases of Src and Janus (JAK) families associate noncovalently with cytoplasmic domains of receptors.

true

True or False: Ligand binding → dimerization → activation of associated tyrosine kinase

true

What does activated kinase phosphorylate?

Tyr on both itself and the receptor

What does tyrosine phosphate do?

phosphorylated Tyr is converted to Tyr and Pi

What are the two domains on receptor tyrosine phosphatase?

• extracellular ligand-binding domain

• intracellular Tyr-phosphatase catalytic domain

What is an example of a receptor tyrosine phosphatase?

CD45 protein

What is CD45? Where is it expressed?

receptor for antibodies; surface of T and B lymphocytes