Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation

Daniel Morgan, Michael Weisenhaus, Sara Shum, Thomas Su, Ruimao Zheng, Chao Zhang, Kevan M. Shokat, Bertil Hille, Donner F. Babcock, G. Stanley McKnight

Research output: Contribution to journalArticle

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Abstract

Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (Cα) of PKA confers sensitivity to the pyrazolo[3,4-d]pyrimidine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the CαM120A mutant protein is expressed and the wild-type Cα is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express CαM120A mutant protein. For CαM120A sperm, 10 μM of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (<90 s) evoked by the HCO3- anion. A continuous (90 min) inhibition with 10 μMof 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in CαM120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional CαM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.

Original languageEnglish (US)
Pages (from-to)20740-20745
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number52
DOIs
StatePublished - Dec 30 2008

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Sperm Capacitation
Cyclic AMP-Dependent Protein Kinases
Spermatozoa
pyrazolo(3,4-d)pyrimidine
Phosphorylation
Mutant Proteins
Tyrosine
Primary Cell Culture
Genetic Recombination
Anions
1-tert-butyl-3-naphthalen-1-ylmethyl-1H-pyrazolo(3,4-d)pyrimidin-4-ylemine
Catalytic Domain
Proteins
Adenosine Triphosphate
Mutation

All Science Journal Classification (ASJC) codes

  • General

Cite this

Morgan, Daniel ; Weisenhaus, Michael ; Shum, Sara ; Su, Thomas ; Zheng, Ruimao ; Zhang, Chao ; Shokat, Kevan M. ; Hille, Bertil ; Babcock, Donner F. ; McKnight, G. Stanley. / Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation. In: Proceedings of the National Academy of Sciences of the United States of America. 2008 ; Vol. 105, No. 52. pp. 20740-20745.
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Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation. / Morgan, Daniel; Weisenhaus, Michael; Shum, Sara; Su, Thomas; Zheng, Ruimao; Zhang, Chao; Shokat, Kevan M.; Hille, Bertil; Babcock, Donner F.; McKnight, G. Stanley.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 52, 30.12.2008, p. 20740-20745.

Research output: Contribution to journalArticle

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T1 - Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation

AU - Morgan, Daniel

AU - Weisenhaus, Michael

AU - Shum, Sara

AU - Su, Thomas

AU - Zheng, Ruimao

AU - Zhang, Chao

AU - Shokat, Kevan M.

AU - Hille, Bertil

AU - Babcock, Donner F.

AU - McKnight, G. Stanley

PY - 2008/12/30

Y1 - 2008/12/30

N2 - Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (Cα) of PKA confers sensitivity to the pyrazolo[3,4-d]pyrimidine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the CαM120A mutant protein is expressed and the wild-type Cα is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express CαM120A mutant protein. For CαM120A sperm, 10 μM of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (<90 s) evoked by the HCO3- anion. A continuous (90 min) inhibition with 10 μMof 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in CαM120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional CαM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.

AB - Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (Cα) of PKA confers sensitivity to the pyrazolo[3,4-d]pyrimidine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the CαM120A mutant protein is expressed and the wild-type Cα is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express CαM120A mutant protein. For CαM120A sperm, 10 μM of 1NM-PP1 prevented PKA-dependent phosphorylation and the activation of motility that are both rapidly (<90 s) evoked by the HCO3- anion. A continuous (90 min) inhibition with 10 μMof 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in CαM120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional CαM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.

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