Rao Lecture 2 January 8

Friday we talked about 1st part of signal transduction. Review: See lecture 1, learning objectives. To review, see slide 2, lecture 1. Strategy is to understand what molecules are involved, time course, where activated. Know what, when, where. Know shared principles slide, including details of GTPase switch proteins and kinases and phosphatases. Know cAMP pathway. Know how glycogen metabolism regulated uses cAMP and PKA. Know the phosphoinositide slide.

Lecture 2:
A few changes have been made since posted lecture. Refer to paper he handed out in class for corrections.
1
Remove objective about PKC. Knowing signal regulation and how signal is brought down is important.
2
Know kinases and phosphatases. Phosphorylation may activate or deactivate an enzyme. Phosphorylating one enzyme activates it, while for another enzyme it inhibits it. In signal transduction, phosphorylation of T,S or Y, can cause binding, or phosphorylating other sites on the same molecule can inhibit binding. Kinases and phosphatases can be regulated by phosphorylation themselves. That’s where we get the kinase cascades.Very little Y phosphorylation, but cancer cells have increased Y phosphorylation.
Relation to receptors:
Receptor itself can have phosphatase or kinase activity (EGF receptor- ligand binding activates).
MAP kinases are soluble, but signaling cascades activate them.
Target proteins:
cSrc can be phosphorylated by many proteins depending on localization within the cell.
FAK is focal adhesion kinase.
3:
History: 1992 Nobel prize was awarded to Krebs and Fischer. Others worked on it before (see list)
Phosphorylase a and b were able to interconvert. Did not know chemical nature of difference, but demonstrated that change in protein played role in regulation. When Fischer and Krebs tried to crystallize phosphorylase, needed ATP and Ca along with Ca dependent kinase to get active form. The forms were interconvertible, and had to be in active form to crystallize. Sutherland incorporated 32P into the enzyme.
4:
Dendogram is of potential kinases in human genome. About 518 of them. DO NOT memorize. All descended from 1 primordial gene.
9 groups:
AGC (named for protein kinases A, G, and C, members of the family)
CAMK calmodulin regulated kinases
CK1 (casein kinase 1)
MAPKK (mitogen activated protein kinase kinase)
CMGC cyclin dependent kinases
PTK- protein tyrosine kinase
Others listed on sheet.

PKC are serine protein kinases. Each isoform has unique domains. RACKs (receptors for activated C kinase) bind to PKC in an isoform-specific way.
5:
Protein phosphatases- two ancestral genes.
About 100 PTPase genes.
Tyr specific- may be receptor-like or intracellular.

PPase- only about 25 genes. About 30% phosphorylated. Resting cell S,T probably heavily phosphorylated.
Phosphatases have catalytic C domain, a is scaffolding protein for translocation, and lots of regulatory domains (50-60 types) B is regulatory domain. Shellfish toxin inhibits PP2A and activates CFTR to cause diarrhea.
6:
Role of protein phosphatases: See slide.
Balance between kinase and phosphatase at low level. Balance shifts when cell is stimulated. Ex:SHP1 mutation leads to disease. Physiological role of SHP1 is to keep inflammatory response suppressed.
PTP1B insulin induces phosphorylation of protein. PTP1B turns off receptor activation. Type II diabetes receptor does not respond properly. Inhibit PTPase- enhance activity of receptor.
PKB and PKC are activated by phosphorylation, so PP2A is essential for homeostasis.

Kinase can be part of receptor. Some have intrinsic phosphatase activity.
Clinical relevance- see slide.
Alteration of normal expression can lead to pathophysiologic state.
7:
Know certain specific pathways. RTK is one well-characterized pathway.
Has 3 domains listed under structure. Cytosolic domain has protein tyrosine kinase activity. Normally, in absence of ligand, is inactive. Binds and induces dimerization. 2 monomeric receptors dimerize. Ligand stabilizes dimeric form. They mutually get activated. Once active, tyrosine kinase phosphorylates tyrosine on intracellular domain of receptor. RTKs mutated in cancer to work without ligand.
8:
Tyrosine kinases phosphorylate many different tyrosines. Each tyrosine has specific amino acid sequences around it. Depending on what signalling molecule binds to receptor, get downstream response through different pathways.
Right- insulin receptor with IRS-1, and example of a multidocking protein. Protein has several accessory binding domains (SH2) to branch into many signaling pathways.
9
RAS is downstream. RAS is a monomeric GTPase switch protein bound to membrane. Activated by GEF. When GDP is bound, it is inactive. GTP makes it active. GEF facilitates exchange. When GTP binds, GTPase activated. Cycle facilitated by GEF and GAP. GAP enhances GTPase activity to deactivate.
How does RAS get activated by RTK?
GRB2 is an adapter protein to bind two proteins together. SH2 domain of GRB2 binds RTK. SH3 domain on GRB2 binds SOS, which is a GEF. Conformational change unmasks RAS binding site on SOS to facilitate exchange of GDP for GTP.
RAS activation is important in human cancers. Mutant RAS is always active. Some cases a mutation prevents GAP binding. No GAP binding- cannot dactivate.
RAS leads to MAPK activation.
10:
How MAPK activated.
RAS activates RAF (a serine/threonine kinase):
Raf inactive in cytosol. Kinase domain is masked there. See slide for rest.
With release of 14-3-3, Raf is activated- serine kinase. Phosphorylates MAPKK (MEK), which can phosphorylate Tyr and Thr. MEK phosphorylates MAPK. MAP kinase activates transcription factors.
11:
A cell only needs a few receptors to produce a cellular response through receptor clustering and signal amplification. Adenylyl cyclase produces several molecules of cAMP. PKA is enzymatic, binds several molecules of substrate, signal amplified severalfold at each step.
12: Termination
Desensitization is seen in GPCRs. PKA can phosphorylate receptor to desensitize. Ligand binds, but no signal results.
Receptors can be endocytosed for recycling or degradation.
Second messengers can be degraded.
Signaling protein can be modified
Counteracting signaling mechanisms can turn off signal as well.
13:
Fine tuning:
Different pathways get different responses from same receptor based on cell type, age, status, and expression of downstream signals.
Many pathways result in same response.
14: Integration and Control
In liver, cells do not normally respond to growth factor. After partial hepatectomy, they do.
Mechanisms:
See slide. Extent of activation or its time course may cause different response.