LKB regulates cdc42 activity in a STRAD dependent pathway.


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Ravi Deevi1, Ishaan Jagan1, Aliya Fatehullah1, Victoria Bingham1, Frederick Charles Campbell1
1Centre for Cancer Research and Cell Biology, Belfast, United Kingdom

Background

The serine/threonine kinase LKB1 acts with cdc42, a master regulator of polarity, to influence tissue morphogenesis. Germ line LKB1 mutation and polarity defects underlie the inherited cancer syndrome, Peutz-jeghers disease. LKB1 mechanisms of cdc42 activation however are unclear. This study tests the hypothesis that STRAD is essential for LKB1 dependent cdc42 activation.

Objectives: are to investigate - (i) LKB1 regulates cdc42 (ii) effects of LKB1 kinase activity on cdc42 activation and (iii) effects of PTEN on LKB1 localization (iv) STRAD activated Cdc42 (v) PTEN effect LKB1 interaction with Cdc42

Method

: HeLa cells which lack LKB1, HCT116 and Caco2 colorectal cells were used. Polarized cell migration was induced by monolayer wounding. Cells were transiently transfected with wild type (wt) LKB1, kinase dead (KD) mutants (LKB1 D176N and K78I) or a mutant which is both kinase dead and remains confined to the nucleus (W308C). Cells were also co-transfected with full length PTEN or PTEN mutants that lack both lipid and protein phosphatise activity and/or full length STRAD?. siRNA knockdown studies were conducted. Protein-protein interactions were assayed by co-immunoprecipitation and cdc42 activation was assessed by GST-PAK pull down assay. Localisation studies were by confocal microscopy and membrane fractionation.

Results

Transient expression of LKB1 and LKB1 kinase dead mutants activated cdc42 in a STRAD-dependent manner.  Conversely, LKB1 siRNA knockdown suppressed cdc42 activity. PTEN interacted with LKB1 and cdc42 in coimmunoprecipitation assays but PTEN did not influence LKB1 localization. PTEN abrogates interactions between LKB1, STRAD and/or cdc42 and impairs STRAD activation of cdc42.

Conclusion

LKB1 activates cdc42 by mechanisms that are independent of LKB1 kinase activity and PTEN.  STRAD can compensate the PTEN in terms of up regulation of Cdc42 activity.