A type 1 phosphoprotein phosphatase active with phosphorylated Mr = 68,000 initiation factor 2 kinase

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Regulation

Background:

• The double-stranded RNA-dependent kinase (PKR) regulates protein synthesis by phosphorylating the alpha subunit of eukaryotic peptide initiation factor 2 (eIF-2).
• PKR activation, triggered by double-stranded RNA, leads to eIF-2 phosphorylation and subsequent inhibition of protein initiation.
• This regulatory mechanism is crucial for translational control.

Purpose of the Study:

• To investigate the role of a type 1 protein phosphatase from reticulocytes in regulating the activity of the double-stranded RNA-dependent kinase.
• To elucidate the mechanism by which this phosphatase affects kinase activity and protein synthesis.

Main Methods:

• Purification and characterization of a type 1 protein phosphatase from reticulocytes.
• Assaying the phosphatase activity on the Mr = 68,000 phosphopeptide of the double-stranded RNA-dependent kinase.
• Measuring kinase activity following phosphatase treatment and re-activation conditions.

Main Results:

• The reticulocyte type 1 protein phosphatase efficiently dephosphorylates the Mr = 68,000 phosphopeptide of the double-stranded RNA-dependent kinase.
• Dephosphorylation directly correlates with a loss of kinase activity.
• Kinase activity can be restored by double-stranded RNA and ATP, indicating cyclic activation-inactivation.
• The phosphatase is Mn2+-dependent and inhibited by inhibitor 2, consistent with Type 1 classification.

Conclusions:

• The type 1 protein phosphatase plays a critical role in inactivating the double-stranded RNA-dependent kinase.
• The phosphorylation state of the Mr = 68,000 peptide is directly linked to the kinase's activation-inactivation cycle.
• Double-stranded RNA is essential for kinase activation and subsequent phosphorylation, but not for maintaining the active state.