1. Calcium levels are tightly regulated in cells, remaining around 10-3 M in the cytosol compared to 10-7 M outside. 2. The plasma membrane uses calcium ATPases and sodium-calcium antiporters to export calcium from the cytosol. Internal membranes like the endoplasmic reticulum also use calcium ATPases to sequester calcium. 3. Receptor tyrosine kinases activate downstream signaling pathways like Ras/Raf/MEK/ERK through phosphorylation of intracellular domains, leading to effects on cell proliferation, survival and other processes.

2. Principes om [Ca2+] laag te houden in de cel (10-3 vs 10-7) Plasma membraan: Na+-driven Ca2+-antiport Ca2+-ATPase Interne membranen ER: Ca2+-ATPase Calcium bindende eiwitten Mitochondrien: actieve import

3. Calcium wave in fertilized egg

4. Calmoduline: een veel gebruikt calcium "receptor"

5. "Dual regulation" van CaCM (calmodulin) kinase

6. Dual regulation of "multisubunit" phosphorylase kinase Complex bevat eigenlijk vier subunits van elk

8. General structure Receptor tyrosine kinase Figure 5.9a The Biology of Cancer (© Garland Science 2007)

9. Many different types of RTKs: different ligand binding sites Figure 5.10 The Biology of Cancer (© Garland Science 2007)

10. Many different types of RTKs: different but overlapping intracellular docking domains Figure 6.9 The Biology of Cancer (© Garland Science 2007)

11. 1. Ligand binding 2. Dimerisatie 3. Activatie kinase 4. Kruisfosforylering General principle of activation Dimerisation + activation

12. Sometimes the kinase is associated

13. Tyrosine gefosforyleerde receptor fungeert als "docking site" voor signalerings enzymen

14. Signaleringsroutes: vorming van eiwit complexen via specifieke eiwit-domeinen

15. Adaptor eiwitten bevatten vaak meerdere “herkenningsdomeinen”

16. Receptor tyrosine kinases maken vaak gebruik van specifieke membraan-gebonden "docking proteins"

17. One of the effects of RTK activation: activation of the G-binding protein Ras 1: RTK bindt complex van Grb2 en Sos 2: Sos is guanine nucleotide exchange factor voor Ras 3: GDP wordt uitgewisseld voor GTP Grb2 Sos

18. Ras, a molecular switch GDP GEF GTP Ras Ras Effector GAP Mutated in tumors vvvvvv GEF: guanine nucleotide exchange factor GAP: GTPase activating protein

19. Mechanism of Ras activation by guanine nucleotide exchange factor

20. Mechanism of Ras inactivation by GTPase activating protein Mechanisme van GAP geinduceerde hydrolyse

21. Visualisation of Ras activation by FRET FRET (fluorescent resonance energy transfer) Mochizuli et al., Nature , 411 (2001) 1066

22. Ras activates Raf-MEK-ERK pathway (a MAP kinase cascade Raf MEK: MAP-ERK kinase MEK ERK: extracellular signal-regulated kinase Also called: MAP kinase ERK

23. Many stimuli can activate Ras by different mechanisms

24. Cycl D1 Ets ERK PLCe Endocytosis Cell cycle control MEK Rab5 Raf1 B-raf NFkB Rin RalBP “Exocist” Stat3 RalGEF Ral JNK Jun PI-3K Cell cycle control Tiam PIP3 AFX FKHR FKHR-L1 p27 p130 PKB Rac PTEN Cytoskeleton GSK3 Survival PDK Bad FasL Bim MnSOD eNOS Ras activates multiple pathways EGF PDGF LPA Insulin TCR Etc. Etc. Etc. Shc R Grb2 Ras SOS

25. PI-3 kinase phosphorylates PIP2 Figure 6.16b The Biology of Cancer (© Garland Science 2007)

26. One of the proteins is PKB (also known as Akt) Fosforylatie door PDK’s maakt PKB actief en vrij Figure 6.19a The Biology of Cancer (© Garland Science 2007)

27. PI3,4,5P3: Translocatie van Protein Kinase B (PKB) en PDK1 (kinase) PDK1: Activatie van Protein Kinase B (PKB)