Protein kinase C-mediated interphase lamin B phosphorylation and solubilization

Area of Science:

• Cell Biology
• Developmental Biology
• Molecular Biology

Background:

• Sperm nuclear envelope disassembly is a critical early event in male pronucleus formation during fertilization.
• The nuclear lamina, a structural component of the nucleus, plays a role in nuclear envelope integrity.

Purpose of the Study:

• To identify the kinase responsible for sperm nuclear lamina disassembly in sea urchin egg cytosol.
• To elucidate the mechanism of lamin B phosphorylation and solubilization during fertilization.

Main Methods:

• Incubation of permeabilized sea urchin sperm nuclei in fertilized egg G1 phase cytosolic extract.
• Phosphorylation assays using specific kinase inhibitors (PKC inhibitors, PKA, p34(cdc2), calmodulin kinase II inhibitors).
• Immunodepletion of cytosolic PKC and restoration with purified rat brain PKC.
• In vitro phosphorylation of sperm lamin B by purified rat brain PKC.
• Two-dimensional phosphopeptide mapping.

Main Results:

• Sperm lamin B undergoes rapid phosphorylation and solubilization in egg cytosol.
• Phosphorylation is dependent on calcium ions (Ca2+) and mediated by protein kinase C (PKC).
• Specific PKC inhibitors blocked phosphorylation, while immunodepletion of PKC abolished it, and purified PKC restored it.
• In vitro studies confirmed sperm lamin B as a substrate for PKC, leading to its solubilization.
• Phosphopeptide maps indicated that the egg cytosolic kinase and purified rat brain PKC are the same.

Conclusions:

• Protein kinase C (PKC) is the primary kinase responsible for the interphase disassembly of the sperm nuclear lamina.
• PKC-mediated phosphorylation of lamin B is a key regulatory event initiating sperm nuclear envelope breakdown during fertilization.