Digital Kinases

A cell model for sensing, integrating and making choice

Communicative & Integrative Biology 3:2, 146-150

Vipul Kumar2012BLZ8552

Aim

• Understanding Cell’s tendency of taking irreversible decisions.(Cell signaling pathways are reversible and graded)

• Applying knowledge to manipulate cellular behavior.(To provide alternate pathways for therapeutic or functional purposes)

• Designing algorithms of functioning of individual cells and multicellular system.(Unraveling the mechanism of functioning of Brain, Differentiation of Cells or Apoptosis etc)

Electronic Circuits

Transistors for:• ON/OFF switches (Bistability)• Amplification of Signals• Regulation of Current flow.• Hysteresis• Two or more “Inputs” with unique “Output”

for a component.

Bistability in Biochemical Pathways

Bistable signal transduction circuits. (a) A double-negative feedback loop. In this circuit, protein A (blue) inhibits or represses B (red), and protein B inhibits or represses A. Thus there could be a stable steady state with A on and B off, or one with B on and A off, but there cannot be a stable steady with both and B on or both A and B off. Such a circuit could toggle between an A-on state and a B-on state in response to trigger stimuli that impinge upon the feedback circuit. (b) A positive feedback loop. In this circuit, A activates B and B activates A. As a result, there could be a stable steady state with both A and B off, or one with both A and B on, but not one with A on and B off or vice versa. Both types of circuits could exhibit persistent, self-perpetuating responses long after the triggering stimulus is removed.

Hysteresis in Biochemical Pathways

Hysteresis and irreversibility in bistable signaling circuits. (a) Hysteresis. Any bistable circuit should exhibit some degree of hysteresis, meaning that different stimulus/response curves are obtained depending upon whether the system began in its off or its on state. (b) Irreversibility. If the feedback in a bistable circuit is sufficiently strong, the circuit may exhibit true irreversibility, so that the system stays in its on state indefinitely after the triggering stimulus is removed.

Kinases: Analogy with ElectronicsCa2+/calmodulin-dependent protein kinase II (CaMKII)• Important mediator of memory and learning.• Role in Ca2+ homeostatis.• Molecular switch (Bistability)• Hysteresis (independent of Ca2+ signal once activated by

autophosphorylation).

MAPK (Mitogen activated Protein Kinase) and JNK (c-Jun N-terminal Kinase)• Ultrasensitivity• Bistability (via positive feedback loop)

**Possibility of sophisticated regulatory network**

Kinases: Analogy with ElectronicsCa2+/calmodulin-dependent protein kinase II (CaMKII)• Important mediator of memory and learning.• Role in Ca2+ homeostatis.• Molecular switch (Bistability)• Hysteresis (independent of Ca2+ signal once activated by

autophosphorylation).

MAPK (Mitogen activated Protein Kinase) and JNK (c-Jun N-terminal Kinase)• Ultrasensitivity• Bistability (via positive feedback loop)

**Possibility of sophisticated regulatory network**

Analogy: Cell as Information Processing Devices

• Particular function/decision made from several inputs.

Apoptosis: An important decision

• Programmed Cell Death• Under irreparable stressed condition.• Irreversible.

Apoptosis: An important decision

AMPK (5’ AMP activated Protein Kinase)• Energy level in cells is tightly regulated to perform various

functions.• Disturbance in Energy level leads to Cell death.• 5’ AMP activated Protein Kinase (AMPK): Energy Sensor.• AMPK is homeostatic regulator of Cell ATP levels.

JNK (c-Jun N-terminal Kinase)• Stress Protein Kinase and regulates death.• Shows “Bistable” system characteristics.

Observations with AMPK and JNK

• Model System: Xenopus oocytes

Two signals were used:• Hyperosmolar Sorbitol• Antimycin

Xenopus is a genus of highly aquatic frogs native to Sub-Saharan Africa.

Observations with AMPK

• AMPK : No Hysteresis Monostable• Requires continuous stimulation• Positive feedback loop not involved.

Observations with JNK

• JNK: Hysteresis Bistable Switch• Positive feedback loop required.

Observations with AMPK and JNK

• Until 2 hrs. the response was analog• At 4 hrs time the response was digital

depending on Stress.• Once crossing the threshold level, the

response was Digital.• Not all cells showed cytochrome-c release.

Complexity of AMPK & JNK in Apoptosis

• In some cases AMPK is found to prevent the apoptosis.

• Sustained activation of AMPK (antimycin) Apoptosis.

• JNK can also have pro or anti-apoptotic role.• Sustained activation of JNKapoptosis• Transitory activation of JNK anti-apoptotic• AMPK and JNK not completely correlated to

cytochrome-c release and caspase-3 activation.

Interpretation of Results

Two possibilities:• AMPK & JNK not involved in cytochrome c

release and another factor responsible for Cell death.

Interpretation of Results

Two possibilities:• AMPK & JNK not involved in cytochrome c

release and another factor responsible for Cell death. (From literature)

• AMPK or JNK digital responses alone are not sufficient to induce Apoptosis. Hence require “combination of digital responses” from different stress sensors (other kinases).

Combination of Digital Responses

• Signal Threshold Level for each Protein Kinase (sensor).

• Only signals crossing threshold level maintain activation. (Hysteresis)

• Signaling threshold for a Kinase may vary for different cell types.

• How many kinases will show digital response to a stimuli?

Combination of Digital Responses

Author’s postulates:• Several Protein Kinases functioning and showing digital or

graded responses.• The combined effect of Kinases: Apoptosis Threshold Level• Relative levels of proapoptotic & antiapoptotic molecules

set this Threshold which needs to be overcome for the irreversible Cell Death Pathway.

• Net protein kinases digitally activated or inhibited can modify pro/anti-apoptotic equilibrium.

Life as a chip: A digital model for cell decisions. Different stimuli (inputs) are sensed by ultrasensitive protein kinases producing a plethora of analog signals, and some of them are converted into digital signals. The digital responses obtained by the network of kinases are integrated and defines a cellular program, which is translated into a cell decision (outputs). Thus, the generation of digital responses by protein kinases might be the basis for important biological processes: from development to memory.

Analog Responses

• All the signals are not completely digital.• Graded response also possible.• Combination of Digital and Analog responses.• Knowledge of all possible pathways and

properties of nodes (Protein Kinases), can give well defined algorithm.

Can this be extended to an Organ: Brain?

Analog Responses

• All the signals are not completely digital.• Graded response also possible.• Combination of Digital and Analog responses.• Knowledge of all possible pathways and

properties of nodes (Protein Kinases), can give well defined algorithm.

Can this be extended to an Organ: Brain?

Brain

• Majority of Protein Kinases expressed in Brain.• These Protein Kinases showing Digital

Responses. (Hypothesis by author)• Physiological concentrations of different

stimuli giving digital responses (in Neurons) for different Kinases.

Brain

• Memory: Long Term Potentiation (LTP).• LTP is activity dependent strengthening of

Synapses.• Individual synapses have all-or-none potentiation

with different Thresholds (i.e. Digital).• LTP regulated by Kinases (cAMP-dependent

protein kinase (PKA), PKC, MAPK, CaMKII and atypical PKC isozyme protein kinase Mzeta (PKMζ))

• Mouse learns to reach the Platform in lesser time (experience).• Does so with aid of visual cue around the perimeter.• Mice with Hippocampus damaged can’t learn.• They can solve if they were trained before brain damage.• Implies that Neurons in Hippocampus necessary for learning.• These newly formed neurons are particularly sensitive to LTP.• Slices from Hippocampus region removed and their CA1 neurons studied

in-vitro with recording electrodes.• Repeated stimulation of presynaptic region of these neurons showed

increased sensitivity (Plasticity).• This plasticity is what we refer as LTP (i.e. long-term strengthening of the

synapses between two neurons).

Brain

• Memory: Long Term Potentiation (LTP).• LTP is activity dependent strengthening of Synapses.• Individual synapses have all-or-none potentiation with

different Thresholds.• Synaptic memories at Hippocampus are encoded in

digital manner.• LTP regulated by Kinases (cAMP-dependent protein

kinase (PKA), PKC, MAPK, Ca2+/calmodulin-dependent protein kinase (CaMKII) and atypical PKC isozyme protein kinase Mzeta (PKMζ))

Brain

Model for Long Term Memory• CaMKII (Digital Kinase) is necessary for LTP in hippocampus.• Can be activated different degrees and hence also able to

function as frequency detector.• CaMKII acts as Bistable switch in postsynaptic density. Which

turns on when a threshold number of kinase sites are phosphorylated.

• Shows Hysteresis for some time due to slow action of Phosphatase.

• PKMζ , acts as constitutive kinase, necessary for LTP maintenance and several long term memories.

Digital Model for Memory• Recall: Retrieval of stored information.• Spike trains (brief sequence of action potentials) generated by

neurons.• Spike trains in analogy with digital signals, representing binary

numbers. Used by brain to make internal calculations.• Neuronal events are digitized via help of Spike Trains. (Hypothesis

by Cesare Marchetti)• Spike train representing digital number sets CaMKII into one of its

1012 potential states. (Freezing of information.)• Spike train carrying same number will reactivate it in terms of

stimulating Calmodulin to liberate Ca2+.• Recall requires switching “ON” of thousands of synapses by a signal.

Digital Model for Memory

• In Brain, there are multitude of such other Kinases (cAMP-dependent protein kinase (PKA), PKC, MAPK, CaMKII and atypical PKC isozyme protein kinase Mzeta (PKMζ)) functioning, at the level of individual cell generating an output.

• All such cells then, contribute to produce a Recall or New Idea as a result of mixed outputs.

Thank You