1. Signal Transduction Pathway Smorgasbord Ron Bose, MD PhD Biochemistry and Molecular Cell Biology Programs Washington University School of Medicine Molecular Cell Biology Lecture. Oct 31, 2013

2. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

3. Resources: Nuclear Hormone Receptors http://www.nursa.org/index.cfm http://www.nursa.org/template.cfm?threadId=113 20 http://www.nursa.org/flash/gene/nuclearreceptor/s tart.html

4. Nuclear Hormone Receptor Superfamily 1.48 Human genes 2.Major Categories: Knock-out in mice causes reproductive, developmental, or metabolic abnormalities. Thyroid Hormone Receptor (TR)- like TR, RAR, PPAR, Vitamin D receptor, LiverX Receptor Estrogen Receptor (ER)- like ER, PR, AR, Estrogen Receptor Related, Glucocorticoid receptor, Mineralocorticoid receptor Retinoid X Receptor (RXR) like RXR, Hepatocyte nuclear factor-4, etc.

5. AF: Activation Function. Mediate transcriptional activation DNA Binding Domain Ligand Binding Domain www.nursa.org/

6. AF: Activation Function. Mediate transcriptional activation www.nursa.org/

7. Bind as homodimers Bind as heterodimers with RXR Hormone response elements are inverted repeats. Hormone response elements are direct repeats.

8. Ligand Present Ligand Absent www.nursa.org/

9. Movie: http://www.nursa.org/flash/gene/nuclearreceptor/start.htmlhttp://www.nursa.org/flash/gene/nuclearreceptor/start.html Tab 12. Nuclear Hormone Action Model www.nursa.org/

10. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

11. Cytokine Receptors – JAK/STAT Pathway Baker et al., Oncogene (2007) 26, 6724–6737

12. Cytokine Receptors Baker et al., Oncogene (2007) 26, 6724–6737

13. JAK = Janus kinases Baker et al., Oncogene (2007) 26, 6724–6737 4 genes in humans and mice TYK2 (first gene in this family to be identified) JAK1, JAK2, JAK3

14. STAT= Signal Transducers and Activators of Transcription Baker et al., Oncogene (2007) 26, 6724–6737

15. Cytokine Receptors – JAK/STAT Pathway Baker et al., Oncogene (2007) 26, 6724–6737

16. from Marmor, Skaria, and Yarden 2004 Recptor Tyrosine Kinases Examples– EGFR, Her2, etc

17. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

18. PI3-kinase – Akt – mTOR Songet al., Nature Rev Mol Cell Bio 2012

19. PI3-kinase – Akt PtdIns(4,5)P 2 (PIP2) PtdIns(3,4,5)P 3 (PIP3) PI3K PTEN Akt PDK1 Zoncu et al., Nature Rev Mol Cell Bio 2011

20. mTOR complexes mTORC1 Rapamycin sensitive Responds to nutrient level, growth factors, energy, and stress. mTORC2 NOT rapamycin sensitive Inputs into mTORC2 less well known. Zoncu et al., Nature Rev Mol Cell Bio 2011

21. mTORC1 substrates S6 kinase 1 (S6K1) eIF-4E binding protein (4E-BP) Zoncu et al., Nature Rev Mol Cell Bio 2011

22. mTORC2 substrates Akt Zoncu et al., Nature Rev Mol Cell Bio 2011 PH domain Kinase Domain PDK1 mTORC2 S473T308 Downstream substrates: TSC complex, PRAS40, etc. 1 3 2

23. Bringing it all together Zoncu et al., Nature Rev Mol Cell Bio 2011 mTOR is a signal integrator, like the chips and circuits in your smart phone

24. Outline 1.Nuclear Hormone Receptors 2.Cytokine Receptors – JAK/STAT Pathway 3.PI3-kinase – Akt – mTOR 4.Regulation of Protein Kinases

25. Manning et al., Science 2002

26. Regulation of Protein Kinases 1.Post-translation modifications.  Phosphorylation-dependent  Activation Loop 2.Protein-protein interactions  Regulatory Subunits  Dimers

27. Structure of PKA catalytic domain C Helix N-lobe C-lobe  Helices  Sheets Caplan, Science STKE 2005

28. Illustration from Nolen et al, Mol. Cell, Vol. 15, p.661-675, 2004 Structural features of the PKA Activation Loop

29. Phosphorylation of the MAP Kinase activation loop Phosphorylation on threonine and tyrosine Phospho-Thr 183 contacts  -C and promotes active conformation Phospho-Thr 183 promotes ERK2 dimerization via conformational changes in C-terminal extension Illustration taken from Huse and Kuriyan, Cell 109, 275-282 (2002) AKT phosphorylation at T308 is also Activation Loop Phosphorylation

30. Unphosphorylated MAP Kinase Structure Thr183 Tyr185 Canagarajah et al Cell 90, 859-869 (1997) Phosphorylated

31. Thr183 Tyr185 Canagarajah et al Cell 90, 859-869 (1997) Unphosphorylated MAP Kinase Structure Phosphorylated

32. Cyclin - Cyclin-dependent kinase (CDK) Complex Cyclin-dependent kinase (Cdk2) Cyclin A Jeffrey et al Nature 376, 313-320 (1995)

33. Cdk2Cdk2.CyclinA Jeffrey et al Nature 376, 313-320 (1995) C-helix Activation Loop

34. Asymmetric Dimer Formed by the EGFR Kinase Domain EGFR kinase domain asymmetric dimer Zhang, Gureasko, Shen, Cole, and Kuriyan. Cell 2006

35. Summary 1.Nuclear hormone receptors consist of a DNA- binding domain and ligand-binding domain. 2.Cytokine receptors signal through the JAK kinases, which have 2 kinase domains, and the STAT transcription factors. 3.mTOR is a signal integrator for metabolic and growth factor signaling. 4.Protein kinases are regulated by PTM’s and protein-protein interactions.