pi5p4k lipid kinase

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

Guanosine Triphosphate (GTP)

Adenine Triphosphate (ATP)

Veronica Hurtado, et. al. 2013

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.

DOWNSTREAM SIGNALLING EVENTS

?Guanosine Triphosphate (GTP)Adenine Triphosphate (ATP)

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

Lemmon, Nature Rev. 2008

Lemmon, Nature Rev. 2008

Lemmon, Nature Rev. 2008

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

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

Identifying PI5P4Ks as a GTP- Sensor Candidate

+ Cell LysateGTP

GTP

MASS SPECTROMETRY

PI5P4Kβ Directly Binds to GTP

Hydrolysis of GTP in vitroReal-time GTP/ATP Hydrolysis in vitro

by PI5P4Kβ

Hydrolysis of GTP in vitro

PI5P4K is a GTP-Dependent Kinase

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 - -

PI5P4Kβ is a GTP-Dependent Kinase

PI5P4Kβ Recognizes Guanine Nucleotides via a‘‘Tetris Spin’’

GMP-PNP AMP-PNP

GTP and ATP analogs are placed in a hydrophobic groove

GTP and ATP analogs are placed in a hydrophobic groove

GTP and ATP analogs are placed in a hydrophobic groove

PI5P4Kβ Recognizes Guanine Nucleotides via a‘‘Tetris Spin’’

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

PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog

PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog

PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog

PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog

PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog

PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog

PI5P4KβT201M and PI5P4KβF205L Specifically Reduce Binding to the GTP Analog

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

Metabolic Adaptation and PI(5)P Accumulation under a GTP-Energy Crisis

B

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

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

GTP-Sensing Activity of PI5P4Kβ Regulates Cell Proliferation

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.

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

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

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

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

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

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

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