A requirement of MAPKAPK2 in the uropod localization of PTEN during FMLP-induced neutrophil chemotaxis q Yue Wu, a Michael O. Hannigan, b Alexey Kotlyarov, c Matthias Gaestel, c Dianqing Wu, b and Chi-Kuang Huang a, * a Department of Pathology, University of Connecticut, Farmington, CT, USA b Department of Genetics and Developmental Biology, University of Connecticut, Farmington, CT, USA c Center for Biochemistry/Institute of Biochemistry, Medial School of Hannover, Hanover, Germany Received 13 October 2003 Abstract The directionality control in chemotaxis is the result of a reciprocal regulation of PI3-kinase and PTEN subcellular localization. MK2 = neutrophils have a directionality loss in fMLP-induced chemotaxis. We found that in polarized WT neutrophils PTEN was localized in the uropod region. However, MK2 = neutrophils or p38 MAPK inhibitor—SB203580—pretreated WT neutrophils showed a disrupted PTEN subcellular localization. Some PTEN was localized at the leading edge of the polarized neutrophils, which may lower the concentration of PI3-kinase lipid product PtdIns(3,4,5)P3 required for directionality sensing. FMLP-stimulated MK2 = neutrophils or SB203580-pretreated WT neutrophils also had disrupted F-actin polarization. F-actin polymerization in- hibitor lantrunculin-B disrupted the polarization of PTEN, but not PtdIns(3,4,5)P3. The results suggest that PTEN uropod polarization is F-actin polymerization-dependent and may be through the effect of MK2 on F-actin polarization. Ó 2004 Elsevier Inc. All rights reserved. Keywords: MK2 = neutrophils; PTEN Phosphatase and tensin homologue (PTEN) was first identified as the tumor suppressor [1,2]. Later PTEN was found to dephosphorylate lipid signaling molecules PtdIns(3,4,5)P3 and PtdIns(3,4)P2 by removing 3- phosphate, and to serve as a key negative regulator of PI3-kinase signaling during chemotaxis [3–5]. Dictyos- telium discoideum cells lacking PTEN have multiple pseudopodia formation and directionality loss in re- sponse to chemoattractant cyclic AMP [6]. PTEN knock-in cells exhibit defects in polarity, motility, and directionality change [7]. PTEN subcellular localization can be regulated by chemotactic stimulation in D. dis- coideum. A fraction of PTEN is bound to membrane in resting cells, whereas PTEN is recruited to the trailing edge of chemotaxing cells after stimulation [6,7]. P38 MAPK pathway is one of the three major mitogen- activated protein-kinase (MAPK) pathways involved in converting extracellular stimulus into numerous intra- cellular responses [8]. MK2 is one of the protein kinases downstream of p38 MAPK [9]. Rapid and transient activation of p38 MAPK pathway was observed in formylated Met–Leu–Phe (fMLP)-stimulated neutrophil chemotaxis [10–12]. Treatment of neutrophils with p38 MAPK inhibitor SB203580 inhibits fMLP-induced che- motaxis [10–12]. Recent studies on the migration of MK2 = mouse neutrophils in Zigmond chambers con- taining fMLP gradients showed that MK2 = neutrophils have chemotaxing directionality loss [13], which suggests that MK2 plays an important role in the regulation of chemotaxis directionality. In this paper, we examined the subcellular localiza- tion of PTEN in WT, SB203580-pretreated WT, and MK2 = neutrophils during fMLP-induced chemo- taxis. We found that PTEN uropod polarization in the chemotaxis is F-actin polymerization-dependent and q Abbreviations: p38 MAPK, p38 mitogen-activated protein kinase; MK2, MAPK-activated protein kinase 2. * Corresponding author. Fax: 1-860-679-2936. E-mail address: huangchi@sun.uchc.edu (C.-K. Huang). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.107 Biochemical and Biophysical Research Communications 316 (2004) 666–672 BBRC www.elsevier.com/locate/ybbrc