Sean S Young | PubMed Health

PubMed2003

Role of integrin-linked kinase in nerve growth factor-stimulated neurite outgrowth.

The role of integrin-linked kinase (ILK), a kinase that is involved in various cellular processes, including adhesion and migration, has not been studied in primary neurons. Using mRNA dot blot and Western blot analysis of ILK in rat and human brain tissue, we found that ILK is expressed in various regions of the CNS. Immunohistochemical and immunocytochemical techniques revealed granular ILK staining that is enriched in neurons and colocalizes with the beta1 integrin subunit. The role of ILK in neurite growth promotion by NGF was studied in rat pheochromocytoma cells and dorsal root ganglion neurons using a pharmacological inhibitor of ILK (KP-392) or after overexpression of dominant-negative ILK (ILK-DN). Both molecular and pharmacological inhibition of ILK activity significantly reduced NGF-induced neurite outgrowth. Survival assays indicate that KP-392-induced suppression of neurite outgrowth occurred in the absence of cell death. ILK kinase activity was stimulated by NGF. NGF-mediated stimulation of phosphorylation of both AKT and the Tau kinase glycogen synthase kinase-3 (GSK-3) was inhibited in the presence of KP-392 and after overexpression of ILK-DN. Consequently, ILK inhibition resulted in an increase in the hyperphosphorylation of Tau, a substrate of GSK-3. Together these findings indicate that ILK is an important effector in NGF-mediated neurite outgrowth.

Animals↗

PubMed2003

Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon.

The protein truncation test (PTT) employs in vitro transcription and translation of amplified cDNA and exonic gDNA to reveal truncating germ-line mutations. In a series of PTT analyses, abnormal splicing in the region encompassing exons 20-23 of BRCA2 was discovered in leucocytes from high-risk breast cancer patients. Although sequencing of the genomic DNA in this region failed to reveal a detectable mutation in these patients, cDNA obtained from this region of BRCA2 uncovered numerous alternative splice isoforms. PTT analysis and nested RT-PCR using RNA from leucocytes from healthy individuals, normal tissue and breast and ovarian cancer tumours demonstrated the presence of these alternatively spliced transcripts in all cases. The splice forms appeared to be more prominent in RNA from aged blood, suggesting that isoform expression was conditional. It is therefore important to distinguish naturally occurring alternative splicing from true splice defects due to mutations when interpreting PTT results.

Alternative Splicing↗