Impaired lymphocyte trafficking in mice deficient in the kinase activity of PKN1

Rana Mashud,Hideyuki Mukai,Kenji Sakimura,Akihide Hayakawa,Takashi Nakayama,Akira Nomachi,Ryosuke Satoh,Koji Kubouchi,Reiko Sugiura,Shigeharu Wakana,Manabu Abe,Takako Hirata,Hiroyuki Ohsaki,Shingo Kamoshida,Sally Danno,Kazuhiko Matsuo

doi:10.1038/s41598-017-07936-9

Abstract

Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain. PKN1[T778A] mutant mice developed to adulthood without apparent external abnormalities, but exhibited lower T and B lymphocyte counts in the peripheral blood than those of wild-type (WT) mice. T and B cell development proceeded in an apparently normal fashion in bone marrow and thymus of PKN1[T778A] mice, however, the number of T and B cell counts were significantly higher in the lymph nodes and spleen of mutant mice in those of WT mice. After transfusion into WT recipients, EGFP-labelled PKN1[T778A] donor lymphocytes were significantly less abundant in the peripheral circulation and more abundant in the spleen and lymph nodes of recipient mice compared with EGFP-labelled WT donor lymphocytes, likely reflecting lymphocyte sequestration in the spleen and lymph nodes in a cell-autonomous fashion. PKN1[T778A] lymphocytes showed significantly lower chemotaxis towards chemokines and sphingosine 1-phosphate (S1P) than WT cells in vitro. The biggest migration defect was observed in response to S1P, which is essential for lymphocyte egress from secondary lymphoid organs. These results reveal a novel role of PKN1 in lymphocyte migration and localization.

Highlights

Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain
It is estimated that ~70% of the body’s lymphocytes are in lymphoid tissues and 2% are in the blood, lymphocytes equivalent to approximately the entire lymphocyte count in the whole body are trafficked between the blood and lymphoid tissues daily[30, 31]
Generated lymphocytes migrate from the bone marrow or thymus into the blood and travel to secondary lymphoid organs, such as the spleen and lymph nodes

Summary

Introduction

Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain. PKN1[T778A] mutant mice developed to adulthood without apparent external abnormalities, but exhibited lower T and B lymphocyte counts in the peripheral blood than those of wild-type (WT) mice. The biggest migration defect was observed in response to S1P, which is essential for lymphocyte egress from secondary lymphoid organs These results reveal a novel role of PKN1 in lymphocyte migration and localization. Yasui et al have reported that PKN1 knockout (KO) mice appear normal and do not exhibit defects in lymphocyte development in PKN1 KO mice within 12 weeks of age[21]. To explore the role of the phosphorylation activity of PKN1 in vivo, we generated knock-in mice expressing kinase-negative mutant of PKN1 by introducing a T778A point mutation in the activation loop of the catalytic domain. We discuss the mechanism underlying this phenomenon and the essential role of PKN1 in lymphocyte trafficking in vivo

Methods

Results

Conclusion