How to analyze the function of kinase substrates
Make a non-phosphorylatable inactive protein:
Make an always phosphorylated protein by
changing to phosphomimetic amino acid
-These approaches are necessary in vitro as well as in vivo.
-In vitro: e.g. incu
General principles III: Signal
Reading for Lecture 3:
Lodish Ch. 15, pp. 632-634;
Ch. 5, pp. 166-167, 209
Ch. 3, p. 90
Ch. 22, 999-1000
Production of second messengers
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Ca2+ can be used as 2nd messenger, because [Ca2+] is kept
very low in cytosol by pumps in the ER and plasma membrane
Ca2+ is a ubiquitous intracellular messenger!
Intracellular Ca2+ after fertilization
Not only GPCRs, but also other receptors, or sperm en
Activation of Ras GTPase
Sos is a Ras-GEF!
2 differences between G and Ras: G is directly activated by
GPCR receptor, G has higher intrinsic GTPase activity
The Ras-GEF Sos opens up Ras-GDP so that GDP can
GTP hydrolysis releases active
How do cells avoid a signaling mess?
1) The cell/tissue-specific developmental history ensures
that only a subset of these proteins is expressed
2) Scaffold proteins can separate common kinases
in synthetic biology
Bashor CJ et al. (2008), Scien
Integrins can directly activate the
Integrin cytoplasmic tail has no
When integrins bind ECM proteins,
Focal adhesion kinase (FAK) is
recruited to FAs
FAK phosphorylates itself, which
recruits Src tyrosine kinase further
Reading for Lecture 9:
Lodish Ch. 15, pp. 656-657; Ch. 16
Homo- or heterodimer
1) A type III (RIII), a proteoglycan, concentrates
ligand at the cell
Usually, cells have to respond to a combination of signals
Integration of signals/cross-regulation
Which signal transduction cascade phosphorylates Smad1?
General principles I
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Reading for Lecture 1:
Lodish Ch. 15, pp. 623-627;
Ch. 3, pp. 92-103
The answers to the questions of the exam are in the lectures
But do read in the textbook pages!
General principles II: Ligandreceptor binding and
molecular analysis of signaling
Reading for Lecture 2:
Lodish Ch. 15, pp. 627-631;
Ch. 3, p. 91
charge to proteins
Cut out bands, renature
and test for activity;