Principles of cell signaling Lecture 4

Transcription

1 Principles of cell signaling Lecture 4 Johan Lennartsson Molecular Cell Biology (1BG320), 2014 Johan.Lennartsson@licr.uu.se 1

2 Receptor tyrosine kinase-induced signal transduction Erk MAP kinase pathway PI3-kinase Akt pathway PLCg PKC pathway 2

3 Phospholipase C hydrolyses PIP 2 into DAG and IP 3 1,2-Diacylglycerol (DAG) and Inositol 1,4,5- trisphosphate (IP 3 ) are potent second messengers Second messengers are intracellular signaling molecules that are not proteins IP 3 binds to and activate IP 3 -receptor in the ER membrane, leading opening of the channel and release of Ca 2+ into the cytoplasm DAG remains in the plasma membrane and functions as a protein kinase C (PKC) activator by facilitating its translocation to the membrane 3

4 PLCg is activated by receptor tyrosine kinases Receptor tyrosine kinases activate PLCg Membrane recruitment depends on both SH2 domain interaction with activated RTK and PH domain interaction with the membrane lipid PIP 3 SH2 PH 4

5 Ca 2+ concentration is kept low in the cytoplasm by Ca 2+ pump proteins Free intracellular [Ca 2+ ] ~ nm. [Ca 2+ ] within lumen of ER ~ mm 5

6 IP 3 promote increased cytoplasmic [Ca 2+ ] Examples of proteins regulated by Ca 2+ Calpain Ca 2+ -dependent protease Troponin Regulator of actin-myosin contraction Calmodulin Intracellular Ca 2+ sensor that can affect activity of many other proteins Synaptotagmin Regulates Ca 2+ -dependent vesicle fusion 6

7 Calmodulin is an intracellular Ca 2+ sensor Upon Ca 2+ binding, calmodulin changes conformation exposing two binding surfaces that participate in protein-protein interactions 7

8 Calmodulin is an intracellular Ca 2+ sensor 4 Ca 2+ Target protein 8

9 Calmodulin-Ca 2+ binding regulates the activity of many signaling proteins Displacement of autoinhinitory domain e.g., CaM kinase II, MLCKs Active site remodeling CaM induced rearragement of thetarget and creates an active site CaM-induced oligomerization e.g., small conductance Ca 2+ - activated K + channel 9

10 1,2-diacylglycerol (DAG) is a membrane associated second messenger 10

11 Protein kinase C family (PKC) PKC:s are a family of serine/threonine kinases The subdivision into subfamilies based on their second messenger requirements for activation Activated downstream of PLC Classical; DAG and Ca 2+ Novel; DAG Atypical 11

12 PKC are translocated to the membrane by their C1 and C2 domain interacting with DAG and Ca 2+, respectively Membrane localization driven by electrostatic and hydrophobic interactions C1 basic Polar head groups DAG hydrophobic Hydrocarbon core DAG 12

13 PKC is activated by translocation from cytoplasm to membrane via displacement of pseudosubstrate C2 C1B C1A Activated Extracellular classical space PKCs are membrane associated 13

14 Examples of pathways involving PKC

15 Review: G-protein coupled receptor activation Lysosomal degradation Internalization (clathrin-coated pits) 15

16 G-protein cycle and regulatory proteins The activity state of a G-protein can be regulated by: GEF (guanine nucleotide exchange factor) GAP (GTPase activating protein) GDI (guanine nucleotide dissociation inhibitor) 16

17 Biochemistry of GTP hydrolysis GTP GDP Energy 17

18 G-protein coupled receptor (GPCR) induced-signal transduction PLCb-PKC pathway camp PKA pathway 18

19 PLCb is activated by G-protein coupled receptors G-protein coupled receptors activate PLCb Membrane recruitment depends on interaction with activated G-proteins (both G a and G bg subunits) 19

20 G-protein coupled receptor (GPCR) induced-signal transduction PLCb-PKC pathway camp PKA pathway 20

21 Activated Ga interact with and stimulate adenylate cyclase activity Adenylate cyclase 21

22 Adenylate cyclase is a large enzyme with 12 transmembrane helices camp is a very important second messenger 22

23 camp is a powerful second messenger camp regulates the function of several effector proteins Protein kinase A (PKA) 23

24 The intracellular concentration of camp must be carefully regulated The balance between adenylate cyclase and campphosphodiesterase determine the intracellular concentration of camp 10 isoforms > 20 isoforms Different adenylate cyclases and camp-phosphodiesterases can be regulated in different allowing for high degree of fine tuning of camp levels 24

25 Adenylate cyclases (AC) can be differentially regulated by different types of G-proteins AC are transmembrane proteins that produce camp from ATP all are activated by G sa -GTP some are inhibited by G ia -GTP some are modulated by G bg, Ca 2+ /calmodulin or kinases (PKC, PKA) Adenylate cyclases function as an integration point of inputs from several hormones 25

26 Smell involves GPCR activation and production of camp that opens an ion channel Membrane depolarization and generation of action potential 26

27 Protein kinase A is activated by camp 27

28 Downstream events provoked by PKA activation: CREB-mediated gene expression PKA can regulate gene expression by phosphorylating CREB Activated PKA can move into the nucleus Inside the nucleus PKA phosphorylates CREB (camp-response element binding protein) Phosphorylated CREB binds to CRE and recruits CBP and activates transcription 28

29 PKA have an important role in regulation of glucose blood levels Glucose storage Can be used for energy production 29

30 PKA have an important role in regulation of glucose blood levels Gluconeogenesis PEPCK 30

31 PKA activation promote glycogen breakdown PKA phosphorylate and thereby inactivate glycogen synthase (this will inhibit glycogen production) PKA phosphorylates and activte glycogen phosphorylase kinase Active glycogen phosphorylase kinase phosphorylates and thereby activate glycogen phosphorylase Active glycogen phosphorylates promoteglycogen degradation (release of glucose 1-phosphate) PKA activation stimulate glykogen breakdown and glucose release into blood stream (in liver cells) 31

32 PKA activation promote glucose synthesis

33 AKAP proteins and regulation of PKA signaling A-kinase anchor proteins (AKAPs) bind PKA and determines its local concentration as well as access to substrates Different AKAPs are target to different intracellular structures Example makap co-localizes PKA and PDE at the nuclear membrane (heart cells) and this result in self-termination of the rise in camp levels (giving only a pulse) 33

34 Different cell types respond differently to PKA activation Different cell types express different subsets of PKA substrates and anchoring proteins (AKAPs) due to the differentiation process. The physiological response to PKA activation depends on which substrates get phosphorylated Epinephrine (adrenalin)-induced PKA activation in different cell types result in: Liver Skeletal muscle Cardiac muscle Adipose tissue Intestinal tissue increased convertion of glycogen to glucose and increased glucose synthesis from amino acids increased conversion of glycogen to glucose increased contraction increased hydrolysis of triglycerides increased fluid secretion 34

35 G-protein coupled receptors can also activate the Erk MAP kinase pathway 35

36 Signal transduction by cytokine receptors Cytokine receptors Receptor tyrosine kinases Stat pathway 36

37 Review: Cytokine receptor activation Cytokine receptor often contain several protein chains 37

38 Signal Transducers and Activators of Transcription (STAT) STAT proteins are latent cytoplasmic transcription factors STAT proteins have to be tyrosine phosphorylated for nuclear localization Stat1, no stimulation Stat1, IFN stimulation Biology of Reproduction (1999) 61:

39 Signal Transducers and Activators of Transcription (STAT) STAT proteins contain SH2-domain and tyrosine phosphorylation site and this allows them to dimerize Stat protein 1 Stat protein 2 SH2 SH2 39

40 STAT signaling pathway 40

41 Activated STAT bind directly to DNA 41

42 STAT signaling pathway Stat1; IFNa/b/g-response Stat2; IFNa/b-response Stat3; several cytokines and growth factors Important for growth regulation Abnormal overactivation of Stat3 correleated with cancer Stat4; IL12-response Stat5a/b; several growth factors and cytokines Regulates expression of milk proteins in breast tissue in response to prolactin Stat6; IL6-response 42

43 TGFb receptor-induced Smad signal transduction Smad pathway 43

44 TGFb receptor activation Figure 14-22a

45 TGFb receptor activation (Serine phosphorylation) TGFb induce clustering of two type I receptors with two type II The type I receptor acts as a substrate for the type II receptor

46 TGFb receptor activation and signaling SARA Figure 14-22c

47 Smad domain structure 47

48 TGFb receptor activation and signaling Figure 14-22d

49 TGFb receptor superfamily consist of TGFb-R, Activin-R and BMP-R 49

50 Receptors and signaling pathways involving proteolysis Wnt pathway involved in many developmental processes critical regulator of stem cells Hedgehog pathway essential for proper embryonic development Notch pathway essential for proper embryonic development regulates cell fate determination 50

51 The Wnt signaling pathway 51

52 Proteins can be marked for degradation by ubiquitin chains (poly-ubiquitination) E1 and E2 activates ubiquitin for attachement to target protein E3 ligase responsible for substrate selection Protein are proteolyticall degraded in the proteasome 52

53 Proteasome is a large complex with multiple protease active sites Selects protein with K48 poly-ubiquitin chains for entry Barrel-like structure with multiple active sites 53

54 Phosphorylation of b-catenin by GSK3b creates a binding site for an E3 ubiquitin ligase E1+E2+E3 complex 54

55 The Wnt signaling pathway 55

56 Role of Wnt signaling for colon epithelium When cell are to far away From Wnt source they stop proliferate Wnt Wnt Wnt Wnt Wnt Wnt Wnt enterocytes : the absorptive cells enteroendocrine cells: hormone-secreting cells goblet cells: mucus-secreting cells Paneth cells: secreting antimicrobial toxins

57 The Hedgehog signaling pathway 57

58 The Notch signaling pathway Proteases g-secretase TACE Presenilin Cell cycle progression (e.g. cyclin D & Myc) Inhibition of apoptosis (e.g. Bcl-2)