EXPERIMENTAL CELL RESEARCH 234, 98–104 (1997) ARTICLE NO. EX973584 The Nuclear Location of Annexin V in the Human Osteosarcoma Cell Line MG-63 Depends on Serum Factors and Tyrosine Kinase Signaling Pathways Javad Mohiti,* Alison M. Caswell,† and John H. Walker* ,1 *Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom; and †Faculty of Health and Social Care, Leeds Metropolitan University, Leeds LS1 3HE, United Kingdom types have been identified where both a calcium signal Serum starvation of MG-63 cells increases their dou- and the activation of MAP kinase are required. Path- bling time from 24 h to 4 days. Cells grown in medium ways leading to the activation of MAP kinase from re- containing 10% fetal calf serum contain high levels of ceptors with tyrosine kinase activity (RTKs) [1] and annexin V in the cell nucleus, whereas growth for 4 from some G-protein-linked receptors have now been days in the absence of serum results in loss of nuclear delineated [2]. annexin V from 72 { 4% of cells. Many of the cells which Many growth stimuli alter the cytosolic free calcium still have nuclear annexin V under these conditions concentration ([Ca 2/ ]i) [3], and the division of some seem to have recently finished dividing. Refeeding types of cell is prevented by inhibition of the calcium cells with medium containing serum restores annexin signal [4]. Both G-protein-linked and tyrosine kinase- V to nuclei within 5 h. Charcoal treatment removes linked receptors can activate pathways leading to the factors from serum that are required to allow annexin generation of a calcium signal, and the calcium can V to return to the nucleus. Protein synthesis is not originate either from intracellular stores in the endo- required for annexin V to return to nuclei since inhibi- plasmic reticulum [5] or in the nuclear envelope [6, 7] tion of protein synthesis with cycloheximide does not or from movement of extracellular calcium across the prevent the serum-induced return of annexin V to nu- plasma membrane. clei. This, and other evidence, indicates that the pres- The calcium signal in activated cells is sensed by two ence of annexin V in nuclei reflects translocation major families of intracellular calcium-binding pro- rather than catabolism and resynthesis. Inhibition of teins. The best characterized is the calmodulin/Tropo- tyrosine kinase activities with genistein attenuates nin C family of E-F hand calcium-binding proteins. Cal- the relocation of annexin V from the cytoplasm to the modulin is clearly involved in the regulation of nuclear nucleus. Thus, the nuclear location of annexin V is con- events and inhibitors of calmodulin can reversibly trolled by signaling pathways involving serum factors block cells in culture in the cell cycle at the G1/S bound- and tyrosine kinases. The results argue for an im- ary and at mitosis [8, 9]. Again, the activation of ki- portant role for annexin V in the cell nucleus.  1997 nases is crucial to these events. Academic Press The second major family of intracellular calcium- binding proteins that mediate the calcium signal are the annexins. These are highly conserved proteins that have been implicated in intracellular calcium handling INTRODUCTION and in the regulation of cell growth [10]. Annexins con- tain a core domain of several repeating sequences each The regulation of cell proliferation by endocrine, of which includes a very highly conserved calcium-bind- paracrine, and autocrine agents which act at the cell ing motif. This corresponds to a loop that projects from surface involves the activation of specific signal trans- the surface of the molecule and may interact with phos- duction pathways, resulting in the altered expression pholipids in activated cells. In addition, annexins pos- of genes that encode proteins involved in cell division. sess a less conserved N-terminal amino acid sequence In many instances it appears that more than one intra- which may be involved in targeting and regulation of cellular signal transduction pathway must be activated annexin activities. A single cell may contain a number to induce proliferation. For example, a number of cell of annexins all present at high concentrations [10 – 12]. Several members of the annexin family have been im- plicated in the regulation of growth processes (see 10 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 01132 333167. E-mail: J.H.Walker@leeds.ac.uk. for review). 98 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.