PDLIM2 (Mystique/SLIM) is a postsynaptic density-95/discs large/zonula occludens-1-Lin-11, Isl-1, Mec-3 (PDZ-LIM) domain protein expressed in the nucleus of T lymphocytes, where it promotes degradation of the p65 subunit of NF-kappaB. It is also expressed at the cytoskeleton in epithelial cells, where it is essential for cell migration. It is not known whether PDLIM2 function at the nucleus and cytoskeleton is linked and whether PDLIM2 subcellular location is regulated in hematopoietic cells. To investigate this, we used the human monocytic leukemia cell line THP-1 that can differentiate into adherent macrophages and the adherent murine macrophage cell line RAW264.7. PMA-induced differentiation of THP-1 cells resulted in increased accumulation of PDLIM2. In differentiated cells, PDLIM2 exhibited retarded mobility indicative of serine phosphorylation, which could be reversed by phosphatases and by inhibition of protein kinase C or ERK kinases. In nondifferentiated THP-1 cells, PDLIM2 was located predominantly in the nucleus, whereas in differentiated cells, PDLIM2 was located predominantly in the cytoplasm. Suppression of PDLIM2 expression in THP-1 and RAW 264.7 cells resulted in decreased adhesion, increased NF-kappaB transcription reporter activity, and increased LPS-induced TNF-alpha production. Overexpression of PDLIM2 in THP-1 cells enhanced cell adhesion. Overall, these findings indicate that PDLIM2 sequestration in the cytoplasm is associated with cell adhesion and increased nuclear activity of NF-kappaB p65. The data suggest that sequestration of PDLIM2 at the cytoskeleton regulates its nuclear function.