1 Lecture 11: Signal transduction and cell cycle regulation Textbook of Receptor Pharmacology, 3rd edition

Announcements! And other important info! Exam Monday! Mentor session Sunday 3- 5 Review session TBA Possible extension on Lab assignment 4 Lab quiz 3: worksheet due Monday in lab, posted on Sakai. 2

Modified from Essential Cell Biology (© Garland Science 2010) If you generated a mutant of Ras that could not hydrolyze GTP  GDP, how would this affect this signaling pathway? 3 A.Increase Ras-induced signaling. B.Decrease Ras-induced signaling. C.Have no effect on Ras-induced signaling. adaptor protein Inactive RasActive Ras Ras GEF Cell surface membrane Active transcription factor

Modified from Essential Cell Biology (© Garland Science 2010) Model: signaling, cyclin, and the restriction point Inactive receptor Inactive transcription factor No G1/S cyclin transcription No G1/S cyclin G1/S cyclin Not dividingDividing Active receptor Signaling molecule G1/S cyclin Active transcription factor Translation of G1/S cyclin Transcription of cyclin Movement through restriction point G1/S-cyclin 4

Modified from Essential Cell Biology (© Garland Science 2010) Combined model: RTK signaling and the restriction point Inactive transcription factor No G1/S cyclin transcription No G1/S cyclin G1/S cyclin Not dividingDividing G1/S cyclin Transcription of G1/S cyclin Nucleus Inactive RTK Active RTK adaptor protein Ras GEF Inactive Ras Ras GEF Inactive RasActive Ras Active transcription factor Inactive MAPKKK Inactive MAPKK Inactive MAPK adaptor protein Translation of G1/S cyclin Movement through restriction point G1/S-cyclin 5

Modified from Essential Cell Biology (© Garland Science 2010) A mutant of Ras that could not hydrolyze GTP  GDP would have what effect on the cell cycle? 6 A.There would be no effect on the cell cycle. B.Cells would arrest at the restriction point. C.Cells would move through the restriction point in the absence of an extracellular signal.

Modified from Essential Cell Biology (© Garland Science 2010) If you expressed a Ras GAP in cells expressing a Ras GTPase mutant, how would this affect the cell cycle? 7 A.There would be no effect on the cell cycle. B.Cells would arrest at the restriction point. C.Cells would move through the restriction point in the absence of an extracellular signal.

G2/M CHECKPOINT G 1 CHECKPOINT or RESTRICTION POINT METAPHASE CHECKPOINT or METAPHASE-TO- ANAPHASE TRANSITION 8 G1/S-cyclin Inactive TF Rb (active) S phase genes S phase regulatory gene expression Active TF Inactive Rb protein S phase genes No S phase regulatory gene expression DNA replication S-cyclin No DNA replication X ✔ Model: Rb, Cdk, and the restriction point

9 An inactivating mutation in Rb would: A.Increase levels of G1/S Cdk activity. B.Increase levels of S cyclin. C.Promote cell cycle arrest at the restriction point. D.Induce movement through the restriction point in the absence of upstream signaling. E.More than one of these is correct.

10 Discussion question: what could be useful strategies to block cell division in cells with an inactivating mutation in Rb? G2/M CHECKPOINT G 1 CHECKPOINT or RESTRICTION POINT METAPHASE CHECKPOINT or METAPHASE-TO-ANAPHASE TRANSITION G1/S-cyclin Inactive TF Rb (active) S phase genes S phase regulatory gene expression Active TF Inactive Rb protein S phase genes No S phase regulatory gene expression DNA replication S-cyclin No DNA replication X ✔

Modified from Essential Cell Biology (© Garland Science 2010) G1/S cyclin Transcription of G1/S cyclin Active RTK Ras GEF Inactive RasActive Ras adaptor protein Translation of G1/S cyclin G1/S-cyclin Combined model: Rb, Cdk, and the restriction point Inactive TF Rb (active) S phase genes Active TF S phase gene expression Inactive Rb S phase genes No S phase gene expression DNA replication S-cyclin No DNA replication X ✔ Transcription of S phase genes (e.g. S cyclin) Translation of S cyclin S-cyclin Nucleus Active transcription factor MAPK cascade

Modified from Essential Cell Biology (© Garland Science 2010) Which of the following is least likely if you expressed a constitutively active RTK and a mutant MAPKK lacking kinase activity? 12 A.Ras would be bound to GTP. B.Rb would be phosphorylated. C.Rb would be active. D.Cell cycle would arrest at the restriction point. E.S Cdk activity would be low.

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