1. Receptor Tyrosine Kinases
Large family of Proteins
Fundamentally different from GPCRs b/c they not only Bind Ligands on the Extracellular side, but also possess a Kinase domain on the Cytosolic side that phosphorylates tyrosines in target proteins
Large family of Proteins
Fundamentally different from GPCRs b/c they not only _____ on the _____ side, but also possess a _______ domain on the ________ side that ___________ in target proteins
a-subunit
Insulin binding domain
b-subunit
Transmembrane helix and kinase domain

2. Receptor Tyrosine Kinases: How do They Work?
Example Protein: Insulin Receptor Tyrosine Kinase (INSR) (ab heterotetramer)
1) Insulin binding to the receptor causes a conformational shift that promotes triple autophosphorylation of the other b-subunit

3. Receptor Tyrosine Kinases: How do They Work?
This Autophosphorylation triggers movement of the Activation Loop which exposes the substrate binding site of the b-subunits
Once activated, INSR binds and phosphorylates Insulin Receptor Subtrate-1 (IRS-1), which then serves as the core for a multiprotein complex

4. Receptor Tyrosine Kinases: How do They Work?
Activation Loop w/ Tyr1158, Tyr1162 and Tyr1163

5. Receptor Tyrosine Kinases: How do They Work?

6. What Happens when INSR Activates IRS-1?
IRS-1 binds to Grb2
Grb2 has an important structural domain: the SH2 domain
Src Homology Domain 2
Binds to Tyr-PO3 residues
Large b-sheet flanked by a-helices
Grb2 is an adapter protein that also has a unique structural domain: the SH3 domain
Src Homology Domain 3
Binds Proline-rich sequences in Tyr-PO3 proteins
X-P-p-X-P motif in substrate where X is a hydrophobic residue in the binding pockets

7. What Happens when INSR Activates IRS-1?
Grb2 binds to Sos via its SH3 domain and Sos binds to the Ras protein which causes it to bind GTP and release GDP
Ras is a G-Protein that is activated by binding GTP. It then binds and actiavtes Raf-1
Raf-1 is a kinase

8. What Happens when INSR Activates IRS-1?
Raf-1 phosphorylates MEK
MEK or MAP Kinase Kinase, phosphorylates ERK which then enters the nucleus and phosphorylates transcription factors responsible for cell division.
Insulin has just stimulated cell division!

9. What Happens when INSR Activates IRS-1?
NOTE: See all the multiple phosphorylation events in the cytoplasmic proteins involved in the pathway?
Why?

10. Crosstalk Between Pathways
Crosstalk is when two pathways have partners or products that interact with each other.
This happens A LOT
The b-adrenergic receptor and INSR tyrosine kinase have such overlap

11. Crosstalk Between Pathways
INSR phosphorylates b-adrenergic receptor at two Tyr in the tail
Protein Kinase B (PKB) is activated by IRS-1 at a branch of the insuling pathway
PKB phophorylates b-adrenergic receptor at two more serines adjacent to the phosphotyrosines

12. Crosstalk Between Pathways
The 4 phophoryl groups on the tail of b-adrenergic receptor trigger internalization of the receptor which desensitizes the cell to epinephrine.
Why would you want this crosstalk to happen in this particular pathway?
What is the end result of the insulin pathway?
What is the end result of the b-adrenergic receptor pathway?

13. 12.11: Cell Cycle Regulation by Protein Kinases
There is a specific family of protein kinases that regualte the life cycle of the cell.
These kinases are controlled by a variety of mechanisms b/c they are so vital in controlling the behaviour of the cell.

14. The Cyclins/CDKs: Protein Kinases of the Cycle
Heterodimeric Protein Kinase Complex
Cyclin: Regulatory subunit, ~ 10 in animal cells
Cyclin Dependent Kinase (CDK): Activated when bound to a cyclin, ~ 8 in animal cells
Without the Cyclin bound: The CDK’s T-loop blocks the active site
When the Cyclin binds, it moves the T-loop out of the way
It is then autophosphorylated on a Thr and the resulting phophoryl-Thr is held in place by a ______

15. The CDKs and Cyclins are Rhythmic
They are controlled by 4 different mechanisms:
Phosphorylation/Dephosphorylation of the T-loop
Degradation of the Cyclin
Oscillating synthesis of both proteins
Specific Protein Inhibitors that bind to the Cyclin/CDK complex
Each of these mechanisms are precise and well-coordinated to keep the cell in check.

16. So how do they work? Well, how ‘bout the G1/S Checkpoint
You don’t want any damage DNA when you are progressing from the Growth 1 (G1) to DNA Synthesis (S) Phase of the Cell Cycle
The E2F transcription factor regulates the genes encoding proteins that are necessary for DNA synthesis
If E2F is active, the DNA will be replicated, damage or not.
CDK2/CyclinE help prevent that…

17. 3) p21 binds to the CDK2/Cyclin E complex and prevents it from functioning (p21 is an inhibitor of the CDK)
1) DNA scanning proteins like ATM or ATR find a break
2) They activate p53 which upregulates expression of p21
4) pRB, a substrate of CDK2, is not phosphorylated which means that it can bind to E2F and prevent it from allowing transcription of genes necessary for DNA synthesis.

18. The Exam
Chapter 7
You’ll have to know the Monosaccharides, Disaccharides, Polysaccharides we discussed
This means you’ll need to know about bonds, structures and biology
Qualitative facts about the glycosaminoglycans
How are sugars attached to proteins?
Why?

19. The Exam Chapter 7 Qualitative facts about Glycolipids Lectins
Nucleotide structure and base pairing (Chargaff’s rules)
DNA structure (Watson and Crick / Rosalind Franklin)

20. The Exam Chapter 8 DNA melting Sanger DNA sequencing Chapter 10
Fatty acids (Chemical structure, names and chemical behaviour)
Classifications of lipids
Chemical structures of triacylglycerols and phospholipids

21. The Exam Chapter 10 Steroid function Chapter 11 Micelles and Bilayers
Fluid Mosaic Model

22. The Exam Chapter 11 Integral and Peripheral membrane proteins
You don’t need to know the 6 classes of transmembrane proteins, just the basic types and details about them
Types of peripheral proteins
Lipid rafts: General composition and role
Caveolin and membrane fusion

23. The Exam Chapter 11 Transport systems across membranes ATPases
Loss of hydration shell, interaction with protein, hydration shell formation; Free energy
Glucose transporter
ATPases
Unique feature of P-type ATPases and how it influences function
V- and F-type ATPases
Roles of ABC transporters

24. The Exam Chapter 12 Key features of all signal transduction systems
GPCRs
Receptor Tyrosine Kinases
Cyclins and CDKs