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Phosphorylation-dependent protein kinase D activation.

R T Waldron¹, T Iglesias, E Rozengurt

¹Department of Medicine, School of Medicine and Molecular Biology, University of California, Los Angeles 90095-1786, USA.

Electrophoresis

|April 10, 1999

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Protein kinase D (PKD) activation is dependent on protein kinase C (PKC) phosphorylation of serine residues 744 and 748. This study demonstrates PKD

Area of Science:

Cellular signaling pathways
Molecular biology
Biochemistry

Background:

Protein kinase D (PKD) is a serine-threonine kinase activated by various stimuli.
PKD activation involves a phosphorylation-dependent mechanism requiring protein kinase C (PKC).
Structural similarities exist between PKD and MEK family kinases, suggesting activation loop phosphorylation is key.

Purpose of the Study:

To investigate the specific serine residues involved in PKD activation.
To elucidate the role of protein kinase C (PKC) in PKD regulation.
To understand the mechanism of PKD activation in response to cellular stimuli.

Main Methods:

Transfection of mutant PKD cDNAs (serine to alanine or glutamic acid) into COS-7 cells.
In vivo 32P-labeling and two-dimensional phosphopeptide mapping of PKD.

Related Experiment Videos

Treatment with PKC inhibitor GFI to assess phosphorylation-dependent activation.

Main Results:

Mutating serines 744 and 748 to alanine abolished PKD activation by phorbol esters.
Mutating these serines to glutamic acid resulted in constitutive PKD activation.
PKC inhibition prevented phorbol ester-induced PKD phosphorylation.
Phosphorylation of serines 744 and 748 is essential for PKD activation.

Conclusions:

PKD activation is directly mediated by PKC-dependent phosphorylation of serines 744 and 748.
These findings confirm PKD's role as a crucial component in PKC signal transduction pathways.
The study provides direct evidence for the in vivo regulation of PKD activity.