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Regulatory domain determinants that control PKD1 activity.

Vitalyi O Rybin1, Jianfen Guo, Erin Harleton

  • 1Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.

The Journal of Biological Chemistry
|May 15, 2012
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Summary

Protein kinase D1 (PKD1) cleavage during apoptosis limits its activity toward protein substrates. This study reveals how specific domains regulate PKD1, impacting cellular responses and suggesting proteolytic events reduce maximal enzyme function.

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Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Enzymology

Background:

  • Protein kinase D1 (PKD1) is activated by growth factors, involving diacylglycerol binding and phosphorylation.
  • PKD1 autophosphorylation at Ser(916) is a marker of its activity.
  • Caspase-3 cleavage of PKD1 during apoptosis generates a fragment whose function is unclear.

Purpose of the Study:

  • To investigate the functional consequences of PKD1 cleavage during apoptosis.
  • To elucidate the distinct roles of regulatory domains in controlling PKD1 activity.
  • To assess the impact of domain deletions on PKD1's catalytic and cellular functions.

Main Methods:

  • Utilized N-terminal deletion mutants (PKD1-Δ1-321) and isolated catalytic domains (PKD1-CD) to model apoptotic fragments.
  • Employed a PH domain deletion mutant (PKD1-ΔPH) to study constitutive activity.
  • Assessed enzyme activity using peptide (CREBtide) and protein substrates (CREB, cTnI).
  • Conducted cell-based assays to evaluate in vivo activation of downstream targets (CREB, ERK).

Main Results:

  • PKD1-Δ1-321 and PKD1-CD showed high Ser(916) autophosphorylation and peptide substrate activity but lacked activation loop phosphorylation and protein substrate activity.
  • PKD1-ΔPH exhibited constitutively high activity toward both peptide and protein substrates.
  • PKD1-Δ1-321 failed to activate CREB and ERK phosphorylation in cell-based studies, unlike wild-type PKD1.
  • Proteolysis removing the C1 domain, but not the PH domain, limits PKD1's maximal activity on protein substrates.

Conclusions:

  • Individual regulatory domains of PKD1 play distinct roles in controlling its activity.
  • Apoptotic cleavage of PKD1, specifically the removal of the C1 domain, impairs its ability to phosphorylate physiological protein substrates.
  • This impairment leads to defective PKD1-dependent cellular responses, highlighting the significance of intact regulatory domains for full enzyme function.