Protein kinases and phosphatases:

1
Protein kinases and phosphatases Role in
natural cryopreservation Justin MacDonald,
PhD CRYO 2007 44th Annual Meeting of the Society
for Cryobiology
2

Signal transduction cold-tolerance...
1. Mammalian Hibernation
Ground Squirrels
2. Vertebrate Freeze Tolerance
Little Brown Bats
Hatchling Painted-Turtles (Chrysemys picta)
Wood frog (Rana sylvatica)
Gray tree frog (Hyla versicolor)
3

Signal transduction cold-tolerance...
3. Invertebrate Freeze Tolerance
Periwinkles (Littorina littorea)
Golden-rod Gall Fly (Epiblema solidaginis)
4
Protein kinases and phosphatases...
The reversible phosphorylation of proteins
regulates nearly every aspect of cell life
The phosphorylation state of a protein may
enhance/suppress its biological
function -increasing/decreasing enzyme
activity -affecting protein-protein
interactions -localization / compartimentalizatio
n -targeting a protein for proteolysis
5
The connectivity of biological studies..
Genome
Alternative splicing, RNA editing, synthesis rates
Transcriptome
Translation rates, mRNA stability,
Post-translational modifications (PTMs)
Proteome
Environmental Factors nutritional
status age disease state
Metabolome
6
Invertebrate freeze-tolerance...
The inter-tidal zone has wide fluctuations in
oxygen availability, salinity, and temperature
Common marine periwinkle (Littorinia littorea
Mollusca, Gastropoda)
Well documented ability to survive both long term
anoxia and freezing exposures. -metabolic rate
depression -selective changes in gene expression
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Signal transduction in marine invertebrates.
Environmental Trigger
Signal Transduction Modules
(PKA, PKC, PKG, PPtases,MAPKs?)
Gene Expression
Metabolic Determinants
(Reversible Protein Phosphorylation)
SARP-19 (EF-hand protein) Metallothionein Ribosoma
l L26 protein KVN protein (novel) Myosin heavy
chain
Anaerobic glycolysis PFK, PK, GP, GS,
PDH Protein Synthesis translation (eIF2)
http//www.carleton.ca/kbstorey
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Periwinkle MAPKs freeze tolerance
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A novel freeze activated MAPK
Gel Filtration Chromatography
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Mitogen-activated protein kinases...
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JNK Activity in Hatchling Turtles
Frozen (0.5h)
Anoxic (20h)
Frozen (1h)
Anoxic (1h)
Anoxic (4h)
Frozen (4h)
Hatchling Turtle
Control
Control
Liver
Liver
Heart
Heart
Kidney
Kidney
Brain
Brain
Greenway Storey (1999) J. Comp. Physiol. B 169
521-7
12

ERK Activity in Hatchling Turtle
Hatchling Turtle Brain
?-MAPK
ERK Activity (pmol/min/mg)
?-pMAPK
Frozen (1h)
Control
Frozen (0.5h)
Frozen (1h)
Frozen (4h)
Control
Anoxic
Greenway Storey (1999) J. Comp. Physiol. B 169
521-7
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PP1 activity in liver of R. sylvatica
PP1 Activity
PP1 Activity
5
10
20
40
50
100
Control
Dehydration
Rehydration
Coordination of freeze-induced cryoprotectant
synthesis PP-1 GPa GS
PP1 Activity
1 h
4 h
12 h
24 h
Control
Anoxia
MacDonald Storey (1999) Cryo-Letters 20, 297-306
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PP-1 in R. sylvatica liver
Anti-PP1c ?
Anti-PP1c ?
Anti-PP1c ??
Cyt cytosol Gly glycogen
Cyt
Gly
Cyt
Gly
Cyt
Gly
Cyt
Gly
Control
Frozen
Control
Frozen
CONTROL
FROZEN
GL
GL
glycogen particle
glycogen particle
PP-1c
PP-1c
GP
P
GP
inactive
active
GS
GS
active
inactive
P
GL - PP1c glycogen targeting subunit
MacDonald Storey (1999) Cryo-Letters 20
297-306.
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Mammalian Hibernation
Seasonal Torpor Pattern of S. richardsonii
35
25
Body Temperature
15
5
0
Oct
Nov
Dec
Jan
Feb
Mar
Apr
Sep
Aug
Physiological Adjustments

• drop in body temperature
• periods of torpor
• reduced heart/ventilatory rate
• metabolic rate depression

Richardsons Ground Squirrel (Spermophilus richar
dsonii)
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Metabolic Adjustments in hibernation

• mRNA synthesis
• protein synthesis
• oxygen consumption
• ATP turnover lt 5 of normal
• overall metabolic rate
• carbohydrate sparing (RQ 0.7)
• (fatty acid oxidation preferred)

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Gene Array Analysis
Analyzing transcriptional patterns in hibernating
animals through heterologous probing of cDNA
arrays
5
-5
Control
Torpid
Recovered
Hierarchical clustering of genes
Storey (2003) Adv. Exp. Med. Biol. 543 21-38.
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Signaling pathway cross-talk.
MAPKKK
MAPKK

MAPK
Targets
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ERK activity in hibernating S. richardsonii...
Hibernating
Euthermic
Plt 0.05, n6
Brain
Kidney
Muscle
Heart
Liver
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
20

ERK protein levels are not altered..
E
E
E
E
E
H
H
H
H
H
Muscle
Liver
Kidney
Brain
Heart
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
21
p42/p44MAPK activity in hibernation
E
E
E
E
E
H
H
H
H
H
ERK 1
ERK 2
Liver
Kidney
Brain
Muscle
Heart
Hibernating
Euthermic
Fold
Fold
Brain
Brain
Muscle
Muscle
Kidney
Kidney
Heart
Heart
Liver
Liver
Plt 0.01, n8
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
22

JNK/SAPK activity in hibernation
E
E
E
H
H
E
E
H
H
H
Liver
Kidney
Brain
Muscle
Heart
Hibernating
Euthermic
Plt 0.01, n8
Brain
Muscle
Kidney
Heart
Liver
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
23

E
E
H
H
E
E
E
H
H
H
c-Jun
Liver
Kidney
Brain
Muscle
Heart
Hibernating
Euthermic
Plt 0.01, n8
Brain
Kidney
Muscle
Heart
Liver
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
24
p38MAPK activity in hibernation
E
E
E
E
H
H
H
H
H
E
phospho-p38MAPK
p38MAPK
Liver
Kidney
Brain
Muscle
Heart
Hibernating
Euthermic
Plt 0.01, n8
Brain
Kidney
Muscle
Heart
Liver
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
25

E
E
H
E
E
E
H
H
H
H
phospho-CREB
CREB
Liver
Kidney
Brain
Muscle
Heart
Hibernating
Euthermic
Plt 0.01, n8
Brain
Kidney
Muscle
Liver
Heart
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
26
MAPK-activated kinases
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MAPK-activated kinases in hibernation
Hibernating
Euthermic
Liver
Kidney
Brain
Muscle
Heart
MacDonald Storey (2005) Int. J. Biochem. Cell
Biol. 37 679-91
28
MAPK-activated protein kinases
p70S6K
MAPKAPK-1
kidney heart
skeletal muscle
P
P
GM
40S ribosomal subunit S6
glycogen particle
PP-1c
GP
TRANSLATIONAL INHIBITION
inactive
GS
active
eEF2 eIF2?
GM -glycogen targeting subunit
CARBOHYDRATE SPARING
Storey Storey (2004) Biol. Rev. Camb. Philos.
Soc. 79 207-33.
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MAPK-activated protein kinases

• Hsp27 (heat shock protein)
• heat osmotic shock resistance
• molecular chaperonin
• F-actin capping activity

MAPKAPK-2
P
Hsp27
? Stabilization of muscle contractile properties
? Myofibrillar reorganization during hibernation
Cardiac myosin light chain (MLC1) gene
Fahlman et al. (2000) Cryobiology 40 332-42.
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Summary

• Signal transduction mechanisms play a central
  role
• in cold-tolerance by coordinating the many
  cellular
• adjustments that are needed for survival
• Complex changes in ERK, JNK, and p38MAPK
  activities
• are observed in hibernation and
  freeze-tolerance
• -stimulate specific gene expression
• -regulation of distinct cellular functions
• (eg, protein synthesis)

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Moraine Lake, Banff National Park
The MACDONALD Lab Meredith Borman Eichiki
Ihara Alexandra Valderrama Janina
Ostrander Elena Edwards
The STOREY Lab Ken Storey Steven
Greenway
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JNK Activity in Hatchling Turtles
JNK Activity ( of Control)
Control
1h
4h
20h
Anoxia Duration
Transcription Factors
Ax
Fz
C
Ax
Fz
C
Liver
Liver
Heart
Heart
Kidney
Kidney
Brain
c-Myc
c-Fos
Greenway Storey (1999) J. Comp. Physiol. B 169
521-7