Torbett, Neil Edward; (2003) Cellular roles of PKN1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Protein Kinase Novel 1 (PKN1), which in part resembles yeast PKCs, has been shown to be under the control of Rho GTPases and 3-Phosphoinositide Dependent Kinase 1. Initial studies tested the hypothesis that Rho-PKN1 inputs control PKB phosphorylation. Despite a demonstrable intervention in Rho function, no evidence was obtained that indicated a role for Rho-PKN1 in PKB control. In seeking a cellular role for PKN1, it was found that GFP tagged PKN1 has the ability to translocate in a reversible manner to a vesicular compartment following hyperosmotic stress. PKN1 kinase activity is not necessary for this translocation and in fact the PKN inhibitor HA1077 is also shown to induce PKN1 vesicle accumulation. PKN1 translocation to these vesicles is dependent on Rac1 (and not Rho) activation although the GTPase binding HR1abc domain is not sufficient for this recruitment. The PKN1 kinase domain however localises constitutively to this compartment and it is demonstrated that this behavior is selective for PKNs. Associated with vesicle recruitment, PKN1 is shown to undergo activation loop phosphorylation and activation. It is established that this activation pathway involves PDK1, which is shown to be recruited to this PKN1 positive compartment upon hyperosmotic stress. Further studies employing PKN1-KO MEF cells place PKN1 upstream of the MKK4-JNK pathway in response to hyperosmotic stress. This PKN1 requirement is shown to be a selective hyperosmotic-induced response and to be specific for JNK, not affecting the closely related ERK1/2 and P38 MAPK pathways. Taken together these findings present a pathway for the selective, hyperosmotic-induced. Rac1 dependent PKN1 translocation, that leads to its endocytosis and PDK1-dependent activation. The role of PKN1 appears to be to facilitate MKK4 activation and its own catalytic activity is indicated to play critical role in this context. This provides a distinctive insight into PKN1 and its specificity of action.

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