Technical Reference #1698

1698.

LOK is a Major ERM Kinase in Resting Lymphocytes and Regulates Cytoskeletal Rearrangement Through ERM Phosphorylation Natalya V. Belkina; Yin Liu; Jian-Jiang Hao; Hajime Karasuyama and Stephen Shaw, National Institutes of Health, PNAS, 106(1698), (2008)
Link To Paper

Abstract:
ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site including unusual preference for tyrosine at P–2. LOK’s activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

Keywords:
ezrin; kinase specificity; knockout; migration; moesin

Materials & Methods:
Immunofluorescence Flow Cytometry and Microscopy. Previously described protocol was used for lymphocyte immunofluorescent staining (7). Murine and Jurkat cells were fixed in suspension by 2% paraformaldehyde for 10 min at 37 °C and then permeabilized BD Perm/Wash Buffer [15 min at room temperature (RT)]. Cells were then washed and stained with fluoresceinconjugated antibody and/or phalloidin for 1 h at RT or overnight at 4 °C followed by 3 washes with BD Perm/Wash Buffer. Cells were analyzed for fluorescence intensity (geometrical mean) on a FACSCalibur. For microscopy cells were allowed to settle on glass-bottom culture dishes no. 1.5 (MatTek) for 10 min. Single-plane images were collected at the midplane of the cell with a Zeiss LSM 510 META confocal microscope using a 100 (N.A. 1.4) oilimmersion objective lens. Polarization was scored blind (7). For scanning electron microscopy cells were fixed processed observed and photographed with the S3000N scanning electron microscope (Hitachi) operated at 10 kV as previously described (7).

Microscopic Technique
Confocal Microscopy, Fluorescence Microscopy

Cell Type(s)
Jurkat