Research Article Nucleocytoplasmic shuttling of STK16 (PKL12), a Golgi-resident serine/ threonine kinase involved in VEGF expression regulation Ba ´rbara Guinea a , Jose ´ Manuel Ligos a , Teresa Laı ´n de Lera a , Juan Martı ´n-Caballero b,1 , Juana Flores c , Manuel Gonzalez de la Pen ˜a a,2 , Javier Garcı ´a-Castro a , Antonio Bernad a, * a Departamento de Inmunologı ´a y Oncologı ´a, Centro Nacional de Biotecnologı ´a/CSIC, Universidad Auto ´noma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain b Servicio de Animalario, Centro Nacional de Biotecnologı ´a/CSIC, Universidad Auto ´noma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain c Departamento de Patologı ´a Animal II, Facultad de Veterinaria, Universidad Complutense de Madrid, E-28040 Madrid, Spain Received 19 May 2005, revised version received 13 September 2005, accepted 7 October 2005 Abstract PKL12/STK16 protein is the first identified mammalian member of a ser/thr kinase subfamily that is conserved across several kingdoms, with a broad expression pattern in murine tissues and cell types. Endogenous STK16 subcellular localization was evaluated by indirect immunofluorescence in NIH/3T3 and NRK cells, demonstrating a Golgi-associated pattern that appears to be independent of signals provided by integrin pathways. When cells were treated with brefeldin A (BFA) or nocodazole, drugs that promote Golgi disorganization, we observed STK16 translocation to the nuclear compartment. Constitutive overexpression of this protein by retroviral vectors also promotes accumulation of STK16 in the nuclear compartment, as shown by subfractionation studies. A kinase-dead STK16 mutant (E202A) was used to demonstrate that both the Golgi association and the nuclear translocation capabilities seem to be independent of the STK16 kinase activity. In addition, we show that STK16 overexpression in several cell lines enhances their capacity to produce and secrete VEGF. To confirm these data in vivo, we injected tumor cells overexpressing STK16 into immunodeficient BALBc/SCID mice. HT1080-derived tumors overexpressing STK16 showed increased volume and number of blood vessels compared to controls. Altogether, these data concur with previous reports suggesting a potential role for STK16 as a transcriptional co-activator. D 2005 Elsevier Inc. All rights reserved. Keywords: ser/thr kinase; Golgi; Translocation; Kinase-dead mutant; Transcriptional activator; VEGF Introduction Protein phosphorylation is a critical regulatory event for intracellular activity and also mediates many responses to external stimuli. Phosphorylation is a rapid reversible modification controlled by specific protein kinases (PK) and phosphatases (PP) in a highly regulated manner [1]. The eukaryotic PK constitute one of the largest families of homologous proteins, currently comprising more than 300 members [2]. They share a 250- to 300-amino-acid catalytic domain implicated in the phosphotransferase reaction [3] and have additional regulatory domains in variable locations with respect to the catalytic domain [1]. PKL12 (PK expressed in day 12 fetal liver) was recently isolated and partially characterized [4]. Various groups have reported the same sequence isolated from several other sources, including Krct, a kinase related to Saccharomyces cerevisiae and Arabidopsis thaliana that was isolated from mouse mammary gland [5], EDPK (embryo-derived PK) from mouse 11-day whole embryo [6], and human MPSK1 (myristoylated and palmitoylated serine – threonine kinase- 1) [7]. All reports correspond to the same mammalian gene, human or murine, for which the denomination STK16 has 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.10.010 * Corresponding author. Fax: +34 91 372 0493. E-mail address: abernad@cnb.uam.es (A. Bernad). 1 Current address: Centro Nacional de Investigaciones Oncolo ´ gicas, Melchor Ferna ´ndez Almagro, E-28029 Madrid, Spain. 2 Current address: Genetrix SL, Marconi 1, Parque Tecnolo ´gico de Madrid, Tres Cantos, E-28760 Madrid, Spain. Experimental Cell Research 312 (2006) 135 – 144 www.elsevier.com/locate/yexcr