Localization of the mixed-lineage kinase DLK/MUK/ZPK to the Golgi apparatus in NIH 3T3 cells

DLK/MUK/ZPK is a serine/threonine kinase that belongs to the mixed-lineage (MLK) subfamily of protein kinases. As is the case for most members of this family, relatively little is known about the physiological role of DLK/MUK /ZPK in mammalian cells. Because analysis of subcellular distribution may p rovide important clues concerning the potential in vivo function of a prote in, an antiserum was generated against the amino terminal region of murine DLK/MUK/ZPK and used for localization studies in wild-type NIH 3T3 cells. L ight microscopic immunocytochemistry experiments performed with the antiser um revealed that DLK/MUK/ZPK was specifically localized in a juxtanuclear s tructure characteristic of the Golgi complex. In support of this, treatment of cells with brefeldin A, a drug known to disintegrate the Golgi apparatu s, caused disruption of DLK/MUK/ZPK perinuclear staining. Ultrastructural o bservation of NIH 3T3 cells also confirmed this localization, showing that most of the immunoreactivity was detected on membranes of the stacked Golgi cisternae. Consistent with localization studies, biochemical analyses reve aled that DLK/MUK/ZPK was predominantly associated with Golgi membranes on fractionation of cellular extracts and was entirely partitioned into the aq ueous phase when membranes were subjected to Triton X-114 extraction. On th e basis of these findings, we suggest that DLK/MUK/ZPK is a peripheral memb rane protein tightly associated with the cytoplasmic face of the Golgi appa ratus.