While patients with advanced prostate cancer initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years. Although hormone-refractory disease is unresponsive to androgen-deprivation, androgen receptor (AR)-regulated signaling pathways remain active and are necessary for cancer progression. Thus, both AR itself and the processes downstream of the receptor remain viable targets for therapeutic intervention. Microarray analysis of multiple clinical cohorts showed that the serine/threonine kinase Ca 2þ /calmodulin-dependent protein kinase kinase b (CaMKKb) is both highly expressed in the prostate and further elevated in prostate cancers. Using cellular models of prostate cancer, we have determined that androgens (a) directly increase the expression of a CaMKKb splice variant and (b) increase functional CaMKKb protein levels as determined by the phosphorylation of both CaMKI and AMP-activated protein kinase (AMPK), two of CaMKKb's primary substrates. Importantly, inhibition of the CaMKKb-AMPK, but not CaMKI, signaling axis in prostate cancer cells by pharmacological inhibitors or siRNA-mediated knockdown blocks androgenmediated migration and invasion. Conversely, overexpression of CaMKKb alone leads to both increased AMPK phosphorylation and cell migration. Given the key roles of CaMKKb and AMPK in the biology of prostate cancer cells, we propose that these enzymes are potential therapeutic targets in prostate cancer. Cancer Res; 71(2); 528-37.Ó2010 AACR.

Nuclear import of classical nuclear localization sequence-containing proteins involves the assembly of an import complex at the cytoplasmic face of the nuclear pore complex (NPC) followed by movement of this complex through the NPC and release of the import substrate into the nuclear interior. This process has historically been thought to require nucleotide hydrolysis as a source of energy. We found, using hydrolysis-resistant GTP analogs and a mutant Ran unable to hydrolyze GTP, that transport of classical nuclear localization sequence containing substrate through the NPC and release of the substrate into the nucleus did not require hydrolysis of GTP by Ran. In fact, for movement of this type of import substrate into the nuclear interior we did not observe a requirement for hydrolysis of any nucleotide triphosphate. We did, however, find that a pool of free GTP (or its structural equivalent) must be added, probably because the GDP Ran that is added must be converted to GTP Ran during the import process. We found that a requirement for GTP hydrolysis can be restored to an import mixture consisting of recombinant import factors by the addition of RCC1, the Ran guanine nucleotide exchange factor.Many nuclear proteins contain a nuclear localization sequence (NLS), 1 the classical type of which consists of either a single or bipartite stretch of primarily basic amino acids. The presence of an NLS on a reporter results in its rapid transport from the cytoplasm through the nuclear pore complex (NPC) and into the nuclear interior. This process can be reconstituted in vitro using cells that have been treated with a low concentration of digitonin, which permeabilizes the plasma membrane to the passage of macromolecules but leaves the nuclear membrane intact (1). Addition of cytosol as a source of import factors, a nucleoside triphosphate (NTP) as a source of energy, and import substrate to these permeabilized cells results in import of the substrate into the nucleus. From this cytosol, four import factors have been identified that together efficiently support nuclear import of a basic NLS-containing substrate in permeabilized cells in the absence of cytosol. The receptor for the classical or basic NLS (karyopherin/importin ␣), together with karyopherin/importin ␤ 1 , targets the import substrate to the NPC (2-8). Thus the addition of these two import factors alone to an in vitro import assay results in binding of import substrate at the nuclear envelope in a characteristic rim pattern. Subsequent passage of import substrate from the NPC into the nuclear interior requires the addition of two other import factors: p10/NTF2 (9, 10) and the small GTPase Ran (11,12). The exact role of the nuclear import factor p10, which preferentially binds GDP-Ran rather than GTP-Ran, has yet to be defined. Ran, however, has been shown to be a key player not only for the nuclear import of a variety of substrates bearing different types of NLSs, but also for the nuclear export of most RNAs and a number of proteins containing different types ...

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

The nuclear import of classical nuclear localization signal-containing proteins depends on importin-a transport receptors. In budding yeast there is a single importin-a gene and in higher eukaryotes there are multiple importin-a-like genes, but in fission yeast there are two: the previously characterized cut15 and the more recently identified imp1. Like other importin-a family members, Imp1p supports nuclear protein import in vitro. In contrast to cut15, imp1 is not essential for viability, but imp1D mutant cells exhibit a telophase delay and mild temperature-sensitive lethality. Differences in the cellular functions that depend on Imp1p and Cut15p indicate that they each have unique physiological roles. They also have common roles because the imp1D and the cut15-85 temperature-sensitive mutations are synthetically lethal; overexpression of cut15 partially suppresses the temperature sensitivity, but not the mitotic delay in imp1D cells; and overexpression of imp1 partially suppresses the mitotic defect in cut15-85 cells but not the loss of viability. Both Imp1p and Cut15p are required for the efficient nuclear import of both an SV40 nuclear localization signal-containing reporter protein and the Pap1p component of the stress response MAP kinase pathway. Imp1p and Cut15p are essential for efficient nuclear protein import in S. pombe.

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoidmodified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.Post-translational modifications of proteins play vital roles in many aspects of cell biology. Hence, identifying and understanding the biological impact of these processes is crucial to furthering our basic understanding of how cells function. Numerous proteins that control important biological regulatory events undergo a complex series of post-translational modifications that are directed by the presence of a so-called CaaX motif at their C terminus. This post-translational pathway, termed protein prenylation, is initiated by the attachment of an isoprenoid lipid to an invariant cysteine residue, the C of the CaaX motif (1, 2). Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is covalently attached to this cysteine by protein farnesyltransferase (FTase) 2 or protein geranylgeranyltransferase-I (GGTase-I), respectively (3). The prenylation step is followed by cleavage of the three C-terminal amino acids (the -AAX) by an endoplasmic reticulum (ER)-bound protease termed Rce1. Finally, the prenylated cysteine, which is now located at the C terminus, is methylated by isoprenylcysteine carboxylmethyltransferase (Icmt), another integral ER membrane protein (4, 5). The final result of these modifications is a protein that contains a prenylated and meth...

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