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Signal transduction 2nd messengers, PIP and Calcium

Classical Phosphatidylinositol pathway. Things to know;What is phospholipase C b, how is it activated and what does it do?What does the second messenger DAG do?What does the second messenger IP3 do?What is protein kinase C and how is it activated?What are phorbol esters and what is the mechanis

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Signal transduction 2nd messengers, PIP and Calcium

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    1. Signal transduction 2nd messengers, PIP and Calcium

    2. Classical Phosphatidylinositol pathway Things to know; What is phospholipase C b, how is it activated and what does it do? What does the second messenger DAG do? What does the second messenger IP3 do? What is protein kinase C and how is it activated? What are phorbol esters and what is the mechanisms behind their role in carcinogenesis?

    5. phosphatidyl inositol 4,5-bisphosphate (PIP2)

    6. Signal transduction through the receptor-triggered hydrolysis of phosphatidyl inositol 4,5-bisphosphate. 1. An activated receptor complex leads to the activation of one of the phospholipase C enzymes. 2. Membrane bound phospholipase C hydrolyzes the phosphodiester bond linking the phosphorylated inositol unit to the acylated glycerol moiety. 3. The hydrolysis of PIP2 leads to the production of 2 intracellular signaling molecules: IP3 and DAG.

    7. G-protein-coupled receptors can activate the inositol phospholipid signaling pathway through Gq.

    8. Phospholipase C (PLC) cytosolic enzymes act on membrane-inserted phosphoinositide substrates. Types: beta and gamma PLC-beta is activated by G-protein-coupled receptors, PLC-gamma is activated by receptor tyrosine kinases.

    9. Phospholipase C cleaves PIP2 to yield two second messengers (DAG and IP3)

    10. The signaling pathway splits into two branches

    11. Model of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) DAG very nonpolar, in contrast to IP3. Remains membrane bound.

    12. Inositol trisphosphate (IP3) couples receptor activation to calcium release from the ER IP3 -rapidly diffuses through the cytoplasm. -induces the rapid release of calcium from intracellular stores-the endoplasmic reticulum and, in smooth muscle cells, the sarcoplasmic reticulum.

    13. Calcium is released through IP3-gated calcium release channels The IP3 gated calcium release channels are regulated by a positive feed back mechanism - calcium binds back to the channels to increase the calcium release. Thus, Ca 2+ release is sudden and ALL or NONE

    14. Two mechanisms terminate the initial calcium response Termination of the response 1.The lifetime of IP3 in a cell is very short: IP3 is rapidly dephosphorylated and inactivated. 2.The calcium that enters the cytosol is rapidly pumped out of the cell. 3. Not all IP3 is dephosphorylated; some is phosphorylated to form inositol 1,3,4,5 tetraphosphate (IP4) which may promote refilling of the intracellular calcium stores.

    15. What does DAG do?

    16. Protein kinase C (PKC) is activated by DAG and Ca++ PKC is a serine/threonine kinase. Activated by DAG and calcium.

    18. PHORBOL ESTERS The importance of protein kinase C in controlling cell division and proliferation is revealed by the action of phorbol esters. phorbol esters -polycyclic alcohol derivatives - carcinogenic (tumor promoters) -activate Protein Kinase C mimic diacylglycerol not readily degradable and the activation is persistent.

    20. Calcium as a signaling molecule Calcium + DAG activates protein kinase C Calcium is used for many signaling purposes in the cell-why? What kinds of proteins bind calcium? What is calmodulin and what does it do? Which family of kinases mediate most of the actions of Calcium in animal cells? Why is CaM-kinase II said to have a memory? What is calcineurin and what does it do? How do the cAMP and Ca++ pathways interact?

    21. Controls on cytosolic Ca ++

    22. We can monitor calcium levels in a cell The concentration of free calcium in a cell can be monitored with fluorescent indicators whose emissions change markedly when calcium is bound (Fura-2 and Fluo-3)

    23. 1. The cytosolic level of Calcium is low in unexcited cells, but can be rapidly raised in response to the appropriate signals. intracellular levels low- phosphate esters are abundant and calcium phosphate is very insoluble. cytosolic level of calcium in unexcited cells is typically 100nm, several orders of magnitude less than the concentration outside the cell. The cytosolic concentration of calcium can be rapidly raised by transiently opening calcium channels in the plasma membrane or in an intracellular membrane.

    24. 2. The binding of calcium to a protein can induce large conformational changes. Calcium binds tightly to proteins-both negatively charged oxygen from glutamate and aspartate and uncharged oxygen from glutamine and asparagine bind well to calcium.

    26. Proteins with EF hand motifs bind calcium. The calcium binding domain is formed by two helices separated by a loop. Calcium binding loop is composed of residues containing side chain oxygen groups The EF hand motif is present in more than 100 known proteins. Example: calmodulin

    27. calmodulin is an EF hand protein that serves as a calcium sensor. Calmodulin Mutlti-functional calcium receptor member of the EF hand family of proteins. consists of two globular lobes joined by a long alpha helix. Each lobe contains two EF hands. There are 4 calcium binding sites (shown as 1, 2, 3, and 4.)

    28. A second view of the structure of calmodulin and the calcium binding loop

    29. Calmodulin is allosterically activated by Calcium Calmodulin activated by the binding of three or four calcium ions. - occurs when the cytosolic calcium level is raised above 500 nm. Not an enzyme Allosteric activator of target proteins. Can serve as a permanent component of an enzyme complex. Calcium

    30. What are calmodulins targets and mechanism of activation? calmodulin targets: many enzymes most important - multifunctional calmodulin-dependent protein kinases (CaM Kinase) and the plasma membrane Calcium-ATPase pump.

    31. Most Ca++ effects are mediated by the CaM-kinases The calmodulin-dependent protein kinases (CaM-kinases): serine/threonine kinases; complex of 12 subunits. Examples: (narrow specificity) 1. myosin light-chain kinase; activates smooth muscle contraction 2. Phosphorylase kinase; activates glycogen breakdown Example of multifunctional CaM kinase; CaM kinase II -found in all cells but enriched in nervous system -Ca++ influx in neurons activates CaM kinase II to phosphorylate tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. -may play a role in memory and learning: mice deficient in the a subunit of CaM kinase II activity cant remember where they put their car keys.

    32. Calmodulin activates CaM-kinases

    33. How? CaM-kinase becomes activated when calmodulin binds to it and remains active after calcium withdrawal.

    34. CaM-kinase has a memory Autophosphorylation: -traps calmodulin so that it does not dissociate from the enzyme until cytosolic calcium levels remain at base line for 10 seconds. -converts the enzyme to a calcium-independent form so that the enzyme remains partially active even after calmodulin dissociates from it. fully active

    35. A protein phosphatase inactivates CaM-kinase CaM-kinase remains partially active until a protein phosphatase removes the phosphate modification

    36. Ways in which the pathways interact: 1. Some forms of cAMP phosphodiesterase and adenylyl cyclase are regulated by Ca++/calmodulin. 2. Share downstream targets. Example: CaM-kinases can phosphorylate CREB at a different site than that phosphorylated by PKA.

    37. Calcineurin: a calcium regulated protein phosphatase. Calcineurin: Protein phosphatase calmodulin binding leads to a conformational change in calcineurin that moves the autoinhibitory domain of calcineurin away from the catalytic site. Calcineurin is a major upstream regulator of NFAT (nuclear factor of activated T-cells).

    38. Role of calcineurin in activation of the NFAT transcription factor NFAT (nuclear factor of activated T-cells) -transcription factor -plays an important role in inducible gene transcription in hematopoietic cells. -NHR a calcineurin-activated regulatory domain that binds calcineurin, is dephosphorylated when calcineurin is activated, and controls the calcineurin-regulated nuclear translocation of NFAT.

    39. Role of calcineurin in NFAT activation There are three steps involved in the activation of NFAT. 1. dephosphorylation 2. nuclear translocation 3. increased affinity for DNA.

    40. Cyclosporin A (CsA) and FK 506 Immunosuppresive drug used in transplant surgery patients. Inhibits calcineurin by blocking the active site of calcineurin and preventing it from acting on any molecules within the cell. -major consequence is the inhibition of NFAT activity, thus preventing the rejection of foreign organs or bone marrow by disrupting the signaling pathway that activates T cells.

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