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

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

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

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

Receptor Tyrosine Kinases: How do They Work?

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

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

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!

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

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

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

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?

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.

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 ______

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.

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…

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.

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?

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

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

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

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

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

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