PI5P4K Lipid Kinase
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Transcript of PI5P4K Lipid Kinase
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Sumita, et al., 2016
BIOCHEMISTRY 641Winter 2016
Supervisor: Dr. MoorheadBrooke Rackel & Ahmad R.
Vahab
The Lipid Kinase PI5P4Kβ Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis
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Guanosine Triphosphate (GTP)
Adenine Triphosphate (ATP)
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Veronica Hurtado, et. al. 2013
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AMPK Senses the Energy Status of the Cell
Increased Glucose Uptake
Glycolysis
Fatty Acid Oxidation
Mitochondrial Biogenesis
Protein Synthesis
Glycogen Synthesis
Gluconeogenesis
Fatty Acid/Cholesterol Synthesis
D. Grahame Hardie, Michael L. J. Ashford. 2014.
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DOWNSTREAM SIGNALLING EVENTS
?Guanosine Triphosphate (GTP)Adenine Triphosphate (ATP)
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Phosphoinositide kinases
• Phospholipid kinases have the ability to catalyze the addition of phosphate groups to the 3-, 4- and/or 5-positions on inositol ring
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Lemmon, Nature Rev. 2008
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Lemmon, Nature Rev. 2008
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Lemmon, Nature Rev. 2008
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Phosphatidylinositol 5-phosphate 4-kinase (PI5P4Ks)
• Mammalian genome encodes 3 isoforms of PtdIns5P 4-Kinase : α, β and γ.
• It is shown that PI5P4Ks have an intrinsic ability to bind to GTP.
• PI5P4K, an emerging target for cancer therapy, controls the levels of lipid second messenger, PI(5)P
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PI5P4Kβ – an Intracellular GTP Sensor
• Acts as a molecular sensor for GTP as it has following three fundamental features:
1. Ability to bind directly to GTP2. appropriate KM value so that its activity is regulated by physiological
changes of the concentration of GTP3. Ability to evoke a signal for cellular functions by regulating PI(5)P
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Identifying PI5P4Ks as a GTP- Sensor Candidate
+ Cell LysateGTP
GTP
MASS SPECTROMETRY
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PI5P4Kβ Directly Binds to GTP
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Hydrolysis of GTP in vitroReal-time GTP/ATP Hydrolysis in vitro
by PI5P4Kβ
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Hydrolysis of GTP in vitro
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PI5P4K is a GTP-Dependent Kinase
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PI5P4K is a GTP-Dependent Kinase
Isoform ATP Km GTP Km ATP Vmax GTP Vmax
PI5P4𝛼 5µM 3µM - -
PI5P4β 236µM 88µM 57µM/min 88µM/min
PI5P4ɣ 31µM 389µM - -
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PI5P4Kβ is a GTP-Dependent Kinase
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PI5P4Kβ Recognizes Guanine Nucleotides via a‘‘Tetris Spin’’
GMP-PNP AMP-PNP
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GTP and ATP analogs are placed in a hydrophobic groove
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GTP and ATP analogs are placed in a hydrophobic groove
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GTP and ATP analogs are placed in a hydrophobic groove
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PI5P4Kβ Recognizes Guanine Nucleotides via a‘‘Tetris Spin’’
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Structure-Based Development of the GTP-Insensitive Mutant of PI5P4Kβ• The crystal structures indicated that Thr-201 (conserved in α & β isoforms) and Phe-205 (conserved in all
3 isoforms) are critical for guanine base recognition in PI5P4Kβ
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PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog
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PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog
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PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog
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PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog
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PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog
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PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog
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PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog
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The GTP-Sensing Activity of PI5P4Kβ Is Required for Metabolic Adaptation
• Treatment with MPA decreased cellular GTP concentration within 4 hr without significantly altering ATP concentration
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Metabolic Adaptation and PI(5)P Accumulation under a GTP-Energy Crisis
B
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Passive Vs. Active Metabolites Passive Metabolites
Log2(MPA(+)/MPA(−)) Log2(MPA(+)/MPA(−))(ionization mode)5-phosphoribosyl-1-pyrophosphate 2.2 1.55Ribose-phosphate 2.12 1.26Inosine 2.1 1.53IMP 1.72 2.07leucine-isoleucine 1.67 0.74Active Metabolites
Log2(MPA(+)/MPA(−)) Log2(MPA(+)/MPA(−))(ionization mode)Hydroxyproline 2.34 –0.06N6-acetyl-l-lysine 2.1 –0.19Citrulline 2.03 0.09Serine 1.9 0.03Threonine 1.89 –0.44
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Biological Significance of the GTP-sensing Activity
• Solid tumor cells need to adapt their metabolism in order to cope with nutrient and energy stresses during tumorigenesis
• PI5P4Ks have been shown to promote tumorigenesis in several types of cancers
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GTP-Sensing Activity of PI5P4Kβ Regulates Cell Proliferation
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Immuno-compromised mice were injected with WT-PI5P4Kβ and PI5P4KβF205L cells, and tumor growth was monitored over an 11-week period.
This result indicates that the GTP-sensing activity of PI5P4Kβ provides an advantage in tumorigenesis in vivo.
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PI5P4Kβ Is A GTP Sensor
• PI5P4Kβ directly binds to GTP• PI5P4Kβ activity is regulated by [GTP] because the Km value
is within physiological GTP levels• PI5P4Kβ changes the level of PI(5)P is response to changes in
[GTP]• GTP- sensing activity is required for metabolic adaptation
and tumorigenesis
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GTP Preference is a Critical biochemical Characteristic
• GTP-dependent kinase activity appears to have evolved from ATP-dependent kinase activity
• Acquisition of GTP preference is not simple
• Need crystal structures of PI5P4K𝛼 and PI5P4Kɣ
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PI5P4Kβ Fills a Gap in the PI5P4K Paradox
• PI5P4K has the 𝛼 highest activity of the isoforms – 100-fold greater than PI5P4Kβ and 2000-fold greater than PI5P4Kβɣ
• PI5P4Kβ knockout mice display dramatic phenotypes
• GTP-sensing activity of PI5P4Kβ could affect PI(5)P levels and use a different signaling pathway than PI5P4K𝛼
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PI5P4Kβ Converts Metabolic Cue from GTP into PI(5)P Signaling
• PI5P4Kβ regulates two lipid second messengers: PI(5)P and PI(4,5)P2.
• Since the majority of PI(4,5)P2 is produced by another pathway by PI4P5K/Type-I PIPKs from PI(4)P,
• It has been suggested that a major role of PI5P4K is to regulate the levels of PI(5)P
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Changes in [PI(5)P] Are Sufficient for Trigger Functional Signaling • with the 85% reduction of GTP concentration, cells harboring the GTP-
insensitive mutant (PI5P4KβF205L) showed no variation of the PI(5)P concentration and significant differences in metabolic responses as compared to WT cells
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The GTP-Sensing Activity of PI5P4Kβ May Be the Achilles Heel for Human Diseases
• PI5P4Kβ knockout mice display the phenotypic link to tumorigenesis as well as whole-body metabolism
• Increased dependence of those pathological states on GTP makes the GTP-sensing activity of PI5P4Kb an Achilles’ heel for those human diseases
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Future Direction
• Potential benefit for pharmaceutical targeting of the GTP-sensing activity of PI5P4Kβ that provides an opportunity to develop unique cancer therapeutics
• Biological cue from GTP concentration need to be integrated into the current energy model as an independent and crucial benchmark for tumorigenesis and metabolic diseases