Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

Increasing evidence suggests functional compartmentalization of interphase nuclei. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions , association of some active genes with nuclear speckles or transcription "factories", and association of transcriptionally repressed genes with heterochromatic regions. Dynamic changes in chromosome compartmentalization imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility, described as "constrained diffusion". None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1-2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1-0.9 microm/min over distances of 1-5 microm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation.

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.