Regulation of PI4,5P2 by MARCKS

1
Regulation of PI(4,5)P2 by MARCKS

• Julia Sable
• Sheetz Lab
• Department of Biological Sciences
• Columbia University

2
Phosphatidylinositol 4,5-bisphosphate (PIP2)
3
PIP2 is important for many cellular processes
Doughman RL et al Spatial Synthesis of PI(4,5)P2
J. Membrane Biol. 194, 77-84 (2003)
4
Biological Functions of PIP2

• PIP2 functions as a potent second messenger by
  regulating membrane cytoskeleton adhesion.
• (Raucher et al Cell 2000)

F-actin
PH

• Visualization of PIP2 by PLC?-PH-GFP shows
  distinct pools of PIP2

5

• Role of talin and PIP2 in focal adhesion
  formation
• Talin recruits the PIP2-synthesizing enzyme
  PIPKI (PIP5K? 661) to adhesions.

(Ling et al, Nature 2002)

• PIP2, in turn, conformationally modulates talin,
  vinculin and - actinin, allowing them to
  interact with other adhesion components.

6
Phosphatidylinositol 4,5-bisphosphate (PIP2)

• Levels of PIP2 are relatively unaltered in most
  cell types and regulation is not well understood.
  
• It has been suggested that a sequestering protein
  may be present that would buffer PIP2 and also
  enable rapid, local release.

• Our Proposed Mechanism of Regulation of PIP2
  Balance Between Synthesis and Sequestration of
  PIP2 through MARCKS protein.

7
Myristoylated alanine-rich protein kinase C
substrate (MARCKS) strongly binds PIP2 in vitro
and in vivo Binds 3 PIP2 with Kd 10-8
M Reversible membrane association upon
Phosphorylation with PKC. (Myrisol-electric
switch mechanism)
ED
Myristoylation
13
PH
8
PIP2
PIP3
9
MARCKS in vivo Translocation by PMA Increases
Free PI(4,5)P2 from the Plasma Membrane.
10
Endogenous MARCKS spatially controls the level of
Free PI(4,5)P2 in the Plasma Membrane
PLC-PH GFP free PIP2
11
Control Experiment with MARCKS Phos- shows both
MARCKS and PKC are required for release of free
PIP2 at the PM.
MARCKS Phos-
PLC PH
merge
Pre
Post
12
Summary

• MARCKS translocation showed an increase of free
  PIP2 at the plasma membrane (as seen by increased
  plasma membrane binding by PLC-PH GFP).
• This indicates that MARCKS can function as a
  polyphosphoinositide buffer to control levels of
  free PIP2 in fibroblasts by forming an
  electro-neutral complex at the plasma membrane.
• Also, the absence of MARCKS rapidly increases
  hydrolysis by PLC.

13
Biochemical Analysis of MARCKS Knockout Lines
Shows 25 PIP2 decrease in turnover rate of
synthesis
Protocol for TLC Exps P32 label MEFs for
4h ChlorMeOH HCL extract--isolate total PI
fraction Run TLC- separate PI isoforms Result
Turnover rate or synthesis of PI over time
25 reduction in turnover of PIP2- inhibition of
PIP5kinase?
14
MARCKS sequestration of PIP2 is important for
actin organization and localization of focal
contacts
a
-/-
/
Actin-mRed
b
-/-
/
PIP5K ?90-GFP
c
Talin FL-GFP
-/-
-/-
/
/
15
Summary

• MARCKS KO cells have significant reduction in
  turnover rates of PIP2.
• (Really New Data) HPLC data also confirms this
  25 reduction in PIP2 mass!
• FRAP shows MARCKS has slow turnover rates in vivo
  indicating long-term sequestration of PIP2 is
  possible.
• MARCKS is required for maintaining adequate PIP2
  levels which in turn regulates the adequate
  formation of focal adhesions and actin
  organization.

16
PKC
(CaCM)
ED
PPases PI5K
PH
PI5K
PH
PH
ED
17
MARCKS (/) TIRF Spreading
(10sec/frame)
Harry Xenias
18
MARCKS (/) TIRF Spreading
-/-
Harry Xenias
19
MARCKS (-/-) TIRF Spreading
Harry Xenias
20
MARCKS (-/-) TIRF Spreading
cell area
Area (?m2)
Harry Xenias
21
MARCKS TIRF Spreading Summary
MARCKS TIRF Spreading Conclusions
22
Acknowledgements Mike Sheetz Sheetz Lab
Members Harry Xenias Song Yi Han Adam Meshel Ana
Kostic Kurt DeVos Analysis Program Ben
Dubin-Thaler Wiggins Lab
23
PIP2 in the formation of Actin Networks.
PIP2 binds to n-WASP to activate it where it can
then bind to Arp2/3 and nucleate new actin
filaments.
24
(No Transcript)