Proteomic techniques were used to identify cardiac proteins from whole heart homogenate and heart mitochondria of Fisher 344/Brown Norway F1 rats, which suffer protein nitration as a consequence of biological aging. Soluble proteins from young (5 mo old) and old (26 mo old) animals were separated by one-and two-dimensional gel electrophoresis. One-and two-dimensional Western blots with an anti-nitrotyrosine antibody show an age-related increase in the immunoresponse of a few specific proteins, which were identified by nanoelectrospray ionization-tandem mass spectrometry (NSI-MS/MS). Complementary proteins were immunoprecipitated with an immobilized anti-nitrotyrosine antibody followed by NSI-MS/MS analysis. A total of 48 proteins were putatively identified. Among the identified proteins were ␣-enolase, ␣-aldolase, desmin, aconitate hydratase, methylmalonate semialdehyde dehydrogenase, 3-ketoacyl-CoA thiolase, acetyl-CoA acetyltransferase, GAPDH, malate dehydrogenase, creatine kinase, electron-transfer flavoprotein, manganese-superoxide dismutase, F1-ATPase, and the voltage-dependent anion channel. Some contaminating blood proteins including transferrin and fibrinogen ␤-chain precursor showed increased levels of nitration as well. MS/MS analysis located nitration at Y105 of the electron-transfer flavoprotein. Among the identified proteins, there are important enzymes responsible for energy production and metabolism as well as proteins involved in the structural integrity of the cells. Our results are consistent with agedependent increased oxidative stress and with free radical-dependent damage of proteins. Possibly the oxidative modifications of the identified proteins contribute to the age-dependent degeneration and functional decline of heart proteins.heart; mitochondria THERE IS INCREASING EVIDENCE for an age-dependent decline of cardiac performance (45). Several studies show this performance decline to be associated with oxidative stress (42, 43, 57, 58), i.e., elevated levels of reactive oxygen species. For example, various biomarkers of oxidative stress such as oxo-2-deoxyguanosine, H 2 O 2 , 3-nitrotyrosine (3-NT), N-⑀-methyllysine, malondialaldehyde, and advanced glycation end products (14,22,47,48,55,62) increase with the age in cardiac tissue. Moreover, dietary restriction significantly reduces the age-dependent accumulation of these oxidative markers in heart, which indicates the importance of oxidative stress in cardiac aging (46).A causal role of reactive oxygen species in the age-dependent decline of cardiac dysfunction can, however, only be defined if biological targets of these species are identified and the physiological impact of biomolecular modification is characterized. A first step to establish molecular mechanisms of age-dependent cardiac dysfunction is the proteomic identification of oxidatively modified proteins. Various studies have indicated the formation of reactive nitrogen species in acute myocardiac disorders such as heart failure (1, 28, 31). These reactive nitrogen species are met...

BackgroundMigration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G1 phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood.Methodology/Principal FindingsIn this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G1 to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G1 to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold) in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16INK4a leading to G1 arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays.Conclusion/SignificanceThe results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear actin might be critical in the regulation of these fundamental endothelial functions.

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-␦ isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-␦-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G 1 -to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.Contraction, migration, and proliferation of vascular endothelial cells are essential features of vascular permeability, endothelial repair after injury, and angiogenesis (1, 2) and are regulated by coordinated changes of actin dynamics (3, 4). The LIMK 1 family of proteins, a member of the class of serine/threonine protein kinases, consists of LIMK1 and LIMK2 that specifically phosphorylate and inactivate cofilin, an actin-depolymerizing protein, thereby regulating actin cytoskeleton rearrangement (5, 6). Other studies showed that cell cycle progression depends on the regulated activity of the LIMK-cofilin system (7,8). The kinase activity of LIMKs is regulated by members of the RhoGTPase family, Rho, Rac, and CdC42, via their downstream protein kinases Rho kinase and p21-activated kinases 1 and 4. These kinases phosphorylate LIMK1 at Thr-508 in the activation loop of the kinase domain (9 -11). Rho kinase activates LIMK2 by phosphorylation at 13).Several lines of evidence suggest that LIMKs also have a function in the nucleus. LIMK1 is predominantly localized in the cytoplasm but accumulates in the nucleus, when the cells are treated with the CRM1-dependent export inhibitor, leptomycin B (LMB) (14). In mouse tissues, various splice forms of LIMK2 have been reported that display an unique cellular localization (15, 16). LIMK2a (full-length) and LIMK2b containing only...

BackgroundThe human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35 (STK35, Clik1) is a member of the NKF 4 (New Kinase Family 4 ) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria.Methodology/Principal FindingsIn this study, we found that the previously published coding sequence of the STK35 gene is incomplete. The newly identified sequence of the STK35 gene codes for a protein of 534 amino acids with a N-terminal elongation of 133 amino acids. It has been designated as STK35L (STK35 long). Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied (endothelial cells, HeLa, and HEK cells) and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes.Conclusions/SignificanceWe found the full-length coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases.