The structures of c-src and v-src

provided an important clue!

Lodish et al. Fig. 24-17

Immuno-precipitation

Normal rabbit serum

Serum from rabbit bearing

src-induced tumor
 Src contains three domains
that are shared with other proteins
Binds polyproline motifs Phosphorylates
other proteins

Binds peptides phosphorylated on Tyr

 Scientists have determined
the precise 3-dimensional structure of Src

Xu et al. Nature. 1997 385:595-602

 Tyrosine phosphorylation of the C-terminus
creates an intramolecular
and inhibitory interaction

What can happen after phosphorylation of Tyr 527 ?

Lodish et al. Fig. 24-17

Structural Motifs:
Myristylated N-terminal "Unique" Domain
SH3 and SH2 Regulatory Domains
Tyrosine Kinase Domain, Activation Loop, Tyr416
Short C-terminal tail, Tyr527
Src Regulation: Autoinhibition:
Ordered Activation "A" Loop forms an alpha-helix, stabilizing the
inactive conformation of the kinase domain.
Blocks peptide-substrate binding site and prevents phosphorylation of
Tyr416.
Where is Src within cells?
This is a covalently attached lipid--what might that mean?
 Identifying The Targets of Src

Western blotting with anti-

phosphotyrosine antibodies
V = v-Src transfected cells
2A/V = non-myristylated v-Src
transfected cells

p120 catenin: modulates cell-

cell adhesion
Reynolds et al. MCB (1989)
 Identifying the targets of Src

Few Examples
- p120 catenin: modulates cell-cell adhesion
- Cortactin A: regulates actin polymerization
- Focal Adhesion Kinase: involved in cell-matrix
interactions

Mike Schaller
 Src modulates both cell-cell
and cell matrix adhesion: The basics

Cell-cell
junctions

Cell-matrix junctions Basal lamina

 Src modulates both cell-cell
and cell matrix adhesion: The basics

Lodish et al. Fig. 22-2

 Epithelial cells secrete a special ECM
called the basal lamina
Epithelial cells

Basal Lamina

Alberts et al. Fig. 19-54

 Cells interact with the ECM
via Focal adhesions, which also anchor
the actin cytoskeleton

Focal
Adhesions
(orange)

Actin: Green Alberts et al. Fig. 17-42

Focal adhesions are
linked to the
actin cytoskeleton

Alberts et al.
Fig. 16-75
A complex network of
proteins links the focal
adhesion to actin and
regulates actin
polymerization

Alberts et al.
Fig. 16-75
 Focal adhesions are sites of
intense protein tyrosine phosphorylation

Focal
adhesions

Actin: Green Phosphotyrosine: Red

 An oversimplified model of Src function
Normal skin cell tightly adherent to ECM

Wounding->platelet recruitment->
cell migration and proliferation

Alberts et al.
 A less oversimplified model
Migratory growth factors
e.g., EGF, PDGF Extracellular matrix

Src
RTKs Integrins
FAK
PI-3-
Adaptors
kinase
Actin
Remodel cell-matrix
From Jones et al. Eur J. Cancer
junctions -> cell motility
36, 1595-1606 (2000)
 FAK is recruited by integrins to the
membrane and is autophosphorylated

- Src binds to
phophorylated FAK

- Src changes conformation

and becomes active

- Src further phosphorylates FAK

- Src-FAK phosphorylate target proteins

Src and FAK act together to regulate other focal
adhesion proteins

Src-FAK signals to regulate adhesion turnover

Src-FAK active = less adhesion, more migration
Src interacting genes

Survival: PI3K, Akt, IKK, NFkB, Caspase 9

Angiogenesis: STAT3, p38 MAPK, VEGF, IL-8

Proliferation: Shc, Grb2/SOS, Ras, Raf, MEK1 / MEK2, Erk1/2

Motility: FAK, p190Rho/GAP, Paxillin, p130CAS, RhoA, JNK

c-jun, MLCK, Myosin
 Scientists have determined
the precise 3-dimensional structure of Src

Active
site
 Scientists have determined
the precise 3-dimensional structure of Src

Active
site
SU6656
 dasatinib

Phase II trials for advanced breast

cancer and advanced sarcomas

Ottmann et al. Blood 110, 2309 (2007)

 Another Src inhibitor is in Phase I/II trials for
metastatic pancreatic, breast, ovarian, and prostate cancers

Active
site
AZD0530

Saracatinib