The BTB domain of the nuclear matrix protein NRP/B is required for neurite outgrowth.
The neuronal nuclear matrix protein, NRP/B, contains a BTB domain and kelch repeats and is expressed in primary neurons but not in primary glial cells. To examine the function of NRP/B in neurons, we analyzed the structure/function of the NRP/B-BTB domain and its role in neurite outgrowth. Based on three-dimensional modeling of NRP/B, we generated an NRP/B-BTB mutant containing three mutations in the conserved amino acids D47A, H60A and R61D that was termed BTB mutant A. BTB mutant A significantly reduced the dimerization of NRP/B compared to wild-type NRP/B. The NRP/B-BTB domain was required for nuclear localization and mediated the association of NRP/B with p110RB through the TR subdomain within the B pocket of p110RB. Overexpression of wild-type NRP/B and NRP/B-BTB domain significantly induced neurite outgrowth in PC12 cells and enhanced the G0-G1 cell population by approximately 23% compared to the control cells, whereas NRP/B-BTB mutant A reduced neurite outgrowth by 70-80%, and inhibited NRP/B-p110RB association. Single cell microinjection of NRP/B-specific antibodies also blocked the neurite outgrowth of PC12 cells upon NGF stimulation. Interference of NRP/B expression by small interfering RNA (NRP/B-siRNA) inhibited neurite outgrowth and suppressed the NGF-induced outgrowth of neurites in PC12 cells. Additionally, p110RB phosphorylation at serine residue 795 was significantly reduced in PC12 cells treated with NRP/B siRNA compared to those treated with control GFP-siRNA, indicating that p110RB is a downstream target of NRP/B. Thus, the BTB domain of NRP/B regulates neurite outgrowth through its interaction with the TR subdomain within the B pocket of p110RB, and the conserved amino acids D47A, H60A and R61D within this domain of NRP/B are crucial residues for neurite extension in neuronal cells. These findings support a role for the BTB-domain of NRP/B as an important regulator of neuronal differentiation.
Kim
Tae-Aug
TA
Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA 02115, USA.
Jiang
Shuxian
S
Seng
Seyha
S
Cha
Kiweon
K
Avraham
Hava Karsenty
HK
Avraham
Shalom
S
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation; D016501:Neurites / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000502:physiology; D009479:Neuropeptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D016716:PC12 Cells; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats
D000906:Antibodies; D004268:DNA-Binding Proteins; D008840:Microfilament Proteins; D009479:Neuropeptides; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C112302:ectodermal-neural cortex 1 protein