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CPNE1-mediated neuronal differentiation can be inhibited by HAX1 expression in HiB5 cells
Hye Young Choi1, Nammi Park2, Boah Lee3
1Department of Radiology, Gyeongsang National University Hospital and College of Medicine, Gyeongsang National University, Jinju, 52727, Republic of Korea.
Insights
The protein HCLS1-related protein X-1 (HAX1) binds to CPNE1, inhibiting CPNE1-induced neuronal differentiation. This interaction, particularly involving HAX1's N-terminal region, affects key neuronal development markers.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- CPNE1 (C1-कश्मीर-नेक्सिन-1) promotes neuronal differentiation in hippocampal progenitor cells.
- Previous studies identified 14-3-3γ and Jab1 as CPNE1 binding proteins regulating this process.
Purpose of the Study:
- To identify novel CPNE1 binding proteins involved in neuronal differentiation.
- To elucidate the role of HCLS1-related protein X-1 (HAX1) in CPNE1-mediated neuronal differentiation.
Main Methods:
- Yeast two-hybrid screening to identify CPNE1 interacting proteins.
- Immunostaining for co-localization analysis.
- In vitro and in vivo interaction assays.
- Overexpression studies in HiB5 cells to assess effects on AKT phosphorylation, Tuj1 expression, and neurite outgrowth.
Main Results:
- HAX1 was identified as a direct binding partner of CPNE1.
- CPNE1 and HAX1 co-localize in the cytosol and plasma membrane.
- CPNE1/HAX1 co-expression reduced AKT phosphorylation, Tuj1 expression, and neurite outgrowth compared to CPNE1 overexpression alone.
- A HAX1 mutant unable to bind CPNE1 did not inhibit CPNE1-mediated neuronal differentiation.
Conclusions:
- HAX1 physically interacts with CPNE1, particularly via its N-terminal region.
- HAX1 acts as a negative regulator of CPNE1-mediated neuronal differentiation.
- The interaction between CPNE1 and HAX1 is crucial for modulating neuronal differentiation processes.
Abstract:
We previously demonstrated that CPNE1 induces neuronal differentiation and identified two binding proteins of CPNE1 (14-3-3γ and Jab1) as potential regulators of CPNE1-mediated neuronal differentiation in hippocampal progenitor cells. To better understand the cellular processes in which CPNE1 participates in neuronal differentiation, we here carried out a yeast two-hybrid screening to find another CPNE1 binding protein. Among the identified proteins, HCLS1-related protein X-1 (HAX1) directly interacts with CPNE1. Immunostaining experiments showed that a fraction of CPNE1 and HAX1 co-localized in the cytosol, particularly in the plasma membrane. In addition, the physical interaction as well as the specific binding regions between CPNE1 and HAX1 were confirmed in vitro and in vivo. Moreover, AKT phosphorylation, Tuj1 (neuronal marker protein) expression, and neurite outgrowth are all reduced in CPNE1/HAX1 overexpressing cells compared to CPNE1 only overexpressing HiB5 cells. Conversely, the HAX1 mutant that does not bind to CPNE1 was unable to inhibit the CPNE1-mediated neuronal differentiation. Together these results indicate that HAX1 is a binding partner of CPNE1 and CPNE1-mediated neuronal differentiation is negatively affected through the binding of HAX1, especially its N-terminal region, with CPNE1.
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