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Title: Characterisation of an alternative splice variant of LKB1
Author: Denison, Fiona Clare
ISNI:       0000 0004 2682 231X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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The LKB1 protein kinase has previously been implicated in a number of major physiological processes including cell proliferation, polarity and energy metabolism. LKB1 is an upstream kinase for AMP-activated protein kinase (AMPK) and 12 members of the AMPK-related family of kinases. In this study, western blotting of LKB1 from various mouse tissues indicated the existence of two molecular weight bands in testis. This, along with the presence of a potential alternative exon at the 3’ end of the gene, led to the discovery that an alternatively-spliced variant of LKB1 is present in mice. This new splice variant has been termed the ‘short form’ (LKB1S) and the original protein termed the ‘long form’ (LKB1L). The proteins are identical in sequence apart from the C-terminus. The overall aim of this study was to characterise the LKB1S protein; its activity, regulation and possible physiological function, using overexpression studies in mammalian cells and analysis of mice lacking LKB1S. An LKB1S-specific antibody was used to show that LKB1S is primarily expressed in testis. It was demonstrated, using overexpression studies, that both forms can activate AMPK, form an active complex with the regulatory proteins STRADα, STRADβ and MO25 and have a similar sub-cellular localisation. The C-terminus of mouse/human LKB1L can be phosphorylated at serine 431/428 (Sapkota et al., 2001). This residue is absent from LKB1S and so the effect of phosphorylation was investigated to determine if this could lead to differences in activity between the splice forms. However, the results suggest that phosphorylation at this site does not affect the ability of LKB1L to activate AMPK in cells. Male mice lacking expression of LKB1S are infertile. The mice display severe sperm abnormalities and sperm counts are significantly reduced. Histological analysis of testis showed that although early spermatogenesis appears to progress normally, abnormalities become apparent later on, culminating in very few spermatozoa reaching the epididymis. One possible reason that has been investigated is that there is a defect in the release of mature sperm from the seminiferous epithelium (spermiation).
Supervisor: Hiscock, Natalie ; Woods, Angela Sponsor: Biotechnology and Biological Sciences Research Council ; Unilever
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral