Conserved Orb6 phosphorylation sites are essential for polarized cell growth in Schizosaccharomyces pombe

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The Ndr-related Orb6 kinase is essential for polarized cell growth in fission yeast.
• The precise mechanism regulating Orb6 activation, particularly the roles of autophosphorylation and upstream kinases, remains largely unelucidated.
• Previous studies indicate Nak1 kinase functions upstream of Orb6 in fission yeast cell polarity pathways.

Purpose of the Study:

• To investigate the mechanism of Orb6 kinase activation and its regulation by Nak1.
• To determine the roles of specific phosphorylation sites (Ser291 and Thr456) in Orb6 activity and complex formation with Nak1.
• To elucidate how Orb6 phosphorylation influences its function in maintaining cell polarity.

Main Methods:

• Overexpression of wild-type and mutant HA-Orb6 constructs (kinase-dead K122A, autophosphorylation site mutant S291A, and phosphorylation site mutant T456A) in fission yeast.
• Coimmunoprecipitation assays to assess Nak1/Orb6 complex formation in vivo.
• In vitro kinase assays using recombinant proteins to evaluate autophosphorylation and Nak1-mediated phosphorylation of Orb6.
• Analysis of cell polarity defects in response to genetic mutations and protein overexpression.

Main Results:

• HA-Orb6 overexpression partially rescued polarity defects in nak1 temperature-sensitive mutants, supporting an upstream role for Nak1.
• Nak1 and Orb6 physically interact in vivo, with the Nak1 C-terminal region necessary for complex formation.
• In vitro kinase assays showed Nak1 did not directly phosphorylate Orb6, suggesting an indirect regulatory mechanism.
• Orb6 autophosphorylation at Ser291 is critical for its function, as the S291A mutant only partially rescued orb6 defects.
• Phosphorylation at Thr456 is essential for Orb6 function in vivo, as the T456A mutant exhibited dominant-negative effects similar to the kinase-dead mutant.
• Both S291A and T456A mutations impaired Nak1/Orb6 coimmunoprecipitation, indicating phosphorylation sites influence complex stability.

Conclusions:

• Orb6 activation involves autophosphorylation at Ser291 and phosphorylation at Thr456 by an upstream kinase.
• These phosphorylation events are crucial for promoting the formation and stability of the Nak1/Orb6 complex.
• The findings propose a model where phosphorylation-dependent complex formation is key to Orb6-mediated regulation of cell polarity in fission yeast.