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In polarized motile cells, stress fibers display specific three-dimensional organization. Ventral stress fibers, attached to focal adhesions at both ends, are restricted to the basal side of the cell and nonprotruding cell sides. Dorsal fibers, transverse actin arcs, and perinuclear actin fibers emanate from protruding cell front toward the nucleus and toward apical side of the cell. Perinuclear cap fibers further extend above the nucleus, associate with nuclear envelope through LINC (linker of nucleoskeleton and cytoskeleton) complex and terminate in focal adhesions at cell rear. How are perinuclear actin fibers formed is poorly understood. We show that the formation of perinuclear actin fibers requires dorsal stress fibers that polymerize from focal adhesions at leading edge, and transverse actin arcs that are interconnected with dorsal fibers in spots rich in α-actinin-1. During cell polarization, the interconnected dorsal fibers and transverse arcs move from leading edge toward dorsal side of the cell. As they move, transverse arcs associate with one end of stress fibers present at nonprotruding cell sides, move them above the nucleus thus forming perinuclear actin fibers. Furthermore, the formation of perinuclear actin fibers induces temporal rotational movement of the nucleus resulting in nuclear reorientation to the direction of migration. These results suggest that the network of dorsal fibers, transverse arcs, and perinuclear fibers transfers mechanical signal between the focal adhesions and nuclear envelope that regulates the nuclear reorientation in polarizing cells. [ABSTRACT FROM AUTHOR]

Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the protooncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5’ UTRs that are implicated in homeostatic adaptation to stress and cancer. [ABSTRACT FROM AUTHOR]

The structural genes, pkg4 and pkg3, encoding two putative protein serine/threonine kinases in Streptomyces granaticolor, have been cloned and sequenced. The genes were isolated after screening genomic sublibraries with specific probes obtained by PCR amplification of chromosomal DNA using degenerate primers which correspond to amino acid sequences highly conserved in eukaryotic protein Ser/Thr kinases. The sequences of these genes predict polypeptide chains of 761 and 780 amino acids for Pkg4 and Pkg3, respectively. The genes are separated by only 2 bp and therefore probably constitute an operon. pkg4, which is positioned upstream of pkg3, contains a UUALeu codon suggesting a developmental-dependent mode of expression. The amino-terminal half of both proteins clearly shares similarities with the family of protein Ser/Thr kinases. Both proteins studied also possess a region rich in Pro and Ala residues and a repeating motif of 11 amino acid residues, the function of which is unknown, in the carboxy-terminal domain. Expression of pkg4 in Escherichia coli gave rise to two different forms : a soluble protein autophosphorylated at threonine residues and an insoluble form phosphorylated at threonine and serine residues. In contrast, when pkg3 was expressed in E. coli, no autophosphorylation was detected either in vivo or in vitro. [ABSTRACT FROM AUTHOR]

[Display omitted] • StkP and GpsB regulate growth and division in Streptococcus pneumoniae. • GpsB directly promotes autophosphorylation of StkP and StkP substrate phosphorylation. • StkP phosphorylates GpsB at multiple Thr and Ser residues. • Phosphorylation of GpsB has a positive effect on StkP activity. • The adaptor GpsB is an essential component of the StkP signaling pathway. StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae , monitors cell wall signals and regulates growth and division in response. In vivo , StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo , with T79 and T83 being the major phosphorylation sites. In vitro , phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo , substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae , ensuring the proper functioning of the StkP signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: The ERK (extracellular signal-regulated kinases) cascade has an evolutionarily conserved three tier architecture consisting of protein kinases Raf, MEK (MAPK/ERK kinase) and ERK. Following activation, ERK phosphorylates various cellular elements leading to diverse cellular responses. Downstream of ERK the family of p90 ribosomal S6 kinases (RSKs) has been proven to be an important conveyor of ERK signaling, however, little is known if ERK and RSK coordinate their functions to generate a specific biological response. Here we show that in epithelial cells conditional activation of the ERK pathway causes phenotypic conversion of epithelial cells to autonomously migrating cells. This process involves two sequential steps characterized by loss of apical–basal polarity followed by cell scattering. The activation of ERK, but not RSK, is sufficient for the execution of the first step and it requires calpain mediated remodeling of actin cytoskeleton. Conversely, RSK regulates the successive stage characterized by cell–cell contact weakening and increased cellular migration. Thus, ERK and RSK regulate different cellular subprograms and coordinated execution of these subprograms in time generates a relevant biological response. Our data also suggest that the mechanism by which the ERK pathway controls a cellular response may be distributed between ERK and RSK, rather than being elicited by a single effector kinase. [Copyright &y& Elsevier]

The spreading of adhering cells is a morphogenetic process during which cells break spherical or radial symmetry and adopt migratory polarity with spatially segregated protruding cell front and non-protruding cell rear. The organization and regulation of these symmetry-breaking events, which are both complex and stochastic, are not fully understood. Here we show that in radially spreading cells, symmetry breaking commences with the development of discrete non-protruding regions characterized by large but sparse focal adhesions and long peripheral actin bundles. Establishment of this non-protruding static region specifies the distally oriented protruding cell front and thus determines the polarity axis and the direction of cell migration. The development of non-protruding regions requires ERK2 and the ERK pathway scaffold protein RACK1. RACK1 promotes adhesion-mediated activation of ERK2 that in turn inhibits p190A-RhoGAP signaling by reducing the peripheral localization of p190A-RhoGAP. We propose that sustained ERK signaling at the prospective cell rear induces p190A-RhoGAP depletion from the cell periphery resulting in peripheral actin bundles and cell rear formation. Since cell adhesion activates both ERK and p190A-RhoGAP signaling this constitutes a spatially confined incoherent feed-forward signaling circuit. [ABSTRACT FROM AUTHOR]

The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis. • ERK2 overexpression leads to partial disruption of cell-cell adhesions. • ERK2 overexpression alone does not induce cell scattering. • Overexpression of ERK2 synergizes with HGF/SF signaling to enhance cell scattering. • Increased expression of ERK2 in pancreatic cancer correlates with a worse prognosis. [ABSTRACT FROM AUTHOR]

Abstract: The establishment of cell polarity is an essential step in the process of cell migration. This process requires precise spatiotemporal coordination of signaling pathways that in most cells create the typical asymmetrical profile of a polarized cell with nucleus located at the cell rear and the microtubule organizing center (MTOC) positioned between the nucleus and the leading edge. During cell polarization, nucleus rearward positioning promotes correct microtubule organizing center localization and thus the establishment of front–rear polarity and directional migration. We found that cell polarization and directional migration require also the reorientation of the nucleus. Nuclear reorientation is manifested as temporally restricted nuclear rotation that aligns the nuclear axis with the axis of cell migration. We also found that nuclear reorientation requires physical connection between the nucleus and cytoskeleton mediated by the LINC (linker of nucleoskeleton and cytoskeleton) complex. Nuclear reorientation is controlled by coordinated activity of lysophosphatidic acid (LPA)-mediated activation of GTPase Rho and the activation of integrin, FAK (focal adhesion kinase), Src, and p190RhoGAP signaling pathway. Integrin signaling is spatially induced at the leading edge as FAK and p190RhoGAP are predominantly activated or localized at this location. We suggest that integrin activation within lamellipodia defines cell front, and subsequent FAK, Src, and p190RhoGAP signaling represents the polarity signal that induces reorientation of the nucleus and thus promotes the establishment of front–rear polarity. [Copyright &y& Elsevier]

Cells attaching to the extracellular matrix spontaneously acquire front–rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front–rear polarity in spreading cells. [ABSTRACT FROM AUTHOR]

Aberrant regulation of the cell cycle is a typical feature of all forms of cancer. In head and neck squamous cell carcinoma (HNSCC), it is often associated with the overexpression of cyclin D1 (CCND1). However, it remains unclear how CCND1 expression changes between tumor and normal tissues and whether human papillomavirus (HPV) affects differential CCND1 expression. Here, we evaluated the expression of D-type cyclins in a cohort of 94 HNSCC patients of which 82 were subjected to whole genome expression profiling of primary tumors and paired normal mucosa. Comparative analysis of paired samples showed that CCND1 was upregulated in 18% of HNSCC tumors. Counterintuitively, CCND1 was downregulated in 23% of carcinomas, more frequently in HPV-positive samples. There was no correlation between the change in D-type cyclin expression and patient survival. Intriguingly, among the tumors with downregulated CCND1, one-third showed an increase in cyclin D2 (CCND2) expression. On the other hand, one-third of tumors with upregulated CCND1 showed a decrease in CCND2. Collectively, we have shown that CCND1 was frequently downregulated in HNSCC tumors. Furthermore, regardless of the HPV status, our data suggested that a change in CCND1 expression was alleviated by a compensatory change in CCND2 expression. [ABSTRACT FROM AUTHOR]