Atypical PKC phosphorylates PAR-1 kinases to regulate localization and activity

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Cellular polarity is crucial for eukaryotic organisms, with Par-1 protein kinases acting as conserved determinants.
• Par-1 regulates asymmetry in organisms like C. elegans and Drosophila, and may influence mammalian epithelial cell polarity.
• Mechanisms regulating Par-1 activity, particularly in mammalian cells, remain largely unknown.

Purpose of the Study:

• To investigate the regulation of human Par-1b (hPar-1b) by atypical protein kinase C (aPKC).
• To identify the specific phosphorylation site on hPar-1b targeted by aPKC and its functional consequences.
• To establish a functional link between aPKC and Par-1 in the context of cellular polarity.

Main Methods:

• Co-immunoprecipitation to assess protein interactions between hPar-1b and aPKC.
• In vitro and in vivo phosphorylation assays to identify and confirm the phosphorylation site (Threonine 595).
• Analysis of hPar-1b kinase activity and plasma membrane localization following phosphorylation.

Main Results:

• Human Par-1b (hPar-1b) interacts with and is negatively regulated by atypical protein kinase C (aPKC).
• hPar-1b is phosphorylated by aPKC at Threonine 595 (T595), a conserved residue across species.
• Phosphorylation of hPar-1b at T595 reduces its kinase activity and plasma membrane localization in vivo.
• The equivalent site in hPar-1a (T564) is also phosphorylated by aPKC.

Conclusions:

• This study reveals a novel regulatory mechanism where aPKC negatively controls Par-1 activity and localization through phosphorylation.
• Establishes a direct functional link between two key regulators of cellular polarity: aPKC and Par-1.
• Proposes a model where aPKC modulates Par-1 function in polarized cells, contributing to the maintenance of cell polarity.