Akt activation disrupts mammary acinar architecture and enhances proliferation in an mTOR-dependent manner

Activation of the serine/threonine kinase Akt/PKB positively impacts on three cellular processes relevant to tumor progression: proliferation, survival, and cell size/growth. Using a three-dimensional culture model of MCF-10A mammary cells, we have examined how Akt influences the morphogenesis of polarized epithelial structures. Activation of a conditionally active variant of Akt elicits large, misshapen structures, which primarily arise from the combined effects of Akt on proliferation and cell size. Importantly, Akt activation amplifies proliferation during the early stages of morphogenesis, but cannot overcome signals suppressing proliferation in late-stage cultures. Akt also cooperates with oncoproteins such as cyclin D1 or HPV E7 to promote proliferation and morphogenesis in the absence of growth factors. Pharmacological inhibition of the Akt effector, mammalian target of rapamycin (mTOR), with rapamycin prevents the morphological disruption elicited by Akt activation, including its effect on cell size and number, and the cooperative effect of Akt on oncogene-driven proliferation, indicating that mTOR function is required for the multiple biological effects of Akt activation during morphogenesis

Immunocytochemical localization of the neuron-specific form of the c-src gene product, pp60c-src(+), in rat brain

Neurons express high levels of a variant form of the c-src gene product, denoted pp60c-src(+), which contains a 6 amino acid insert in the amino-terminal half of the c-src protein. We have determined the localization of pp60c-src(+) in neurons using an affinity-purified anti-peptide antibody, referred to as affi-SB12, that exclusively recognizes this neuron-specific form of the c-src gene product. Using affi-SB12, we examined the distribution of pp60c-src(+) by immunoperoxidase staining of sections through adult rat brains, pp60c-src(+) was widely distributed in rat brain and appeared to be differentially expressed in subpopulations of neurons. The majority of immunoreactive neurons was found in the mesencephalon, cerebellum, pons, and medulla. Telencephalic structures that contained substantial populations of pp60c-src(+)-immunoreactive neurons included layer V of the cerebral cortex and the ventral pallidum. Within individual neurons, pp60c-src(+) immunoreactivity was localized to the cell soma and dendritic processes, while labeling of axons and nerve terminals (puncta) was not as readily detected. Dense accumulations of immunoreactive axons were rare, being most prominent in portions of the inferior and superior olive, and in the spinal trigeminal nucleus. While the regional distribution of pp60c-src(+) immunoreactivity does not correlate with any specific neuronal cell type or first messenger system, this unique pattern of expression of pp60c-src(+) suggests the existence of a previously uncharacterized functional organization within the brain. Furthermore, the localization of this neuron-specific tyrosine kinase in functionally important areas of the nerve cell, namely, dendritic processes, axons, and nerve terminals, suggests that pp60c-src(+) may regulate pleiotropic functions in specific classes of neurons in the adult central nervous system

Role for polo-like kinase 4 in mediation of cytokinesis.

The mitotic protein polo-like kinase 4 (PLK4) plays a critical role in centrosome duplication for cell division. By using immunofluorescence, we confirm that PLK4 is localized to centrosomes. In addition, we find that phospho-PLK4 (pPLK4) is cleaved and distributed to kinetochores (metaphase and anaphase), spindle midzone/cleavage furrow (anaphase and telophase), and midbody (cytokinesis) during cell division in immortalized epithelial cells as well as breast, ovarian, and colorectal cancer cells. The distribution of pPLK4 midzone/cleavage furrow and midbody positions pPLK4 to play a functional role in cytokinesis. Indeed, we found that inhibition of PLK4 kinase activity with a small-molecule inhibitor, CFI-400945, prevents translocation to the spindle midzone/cleavage furrow and prevents cellular abscission, leading to the generation of cells with polyploidy, increased numbers of duplicated centrosomes, and vulnerability to anaphase or mitotic catastrophe. The regulatory role of PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers

The behavior of osteoblast-like cells on various substrates with functional blocking of integrin-β1 and integrin-β3

This study was designed to examine the influence of integrin subunit-β1 and subunit-β3 on the behavior of primary osteoblast-like cells, cultured on calcium phosphate (CaP)-coated and non coated titanium (Ti). Osteoblast-like cells were incubated with specific monoclonal antibodies against integrin-β1 and integrin-β3 to block the integrin function. Subsequently, cells were seeded on Ti discs, either non coated or provided with a 2 μm carbonated hydroxyapatite coating using Electrostatic Spray Deposition. Results showed that on CaP coatings, cellular attachment was decreased after a pre-treatment with either anti-integrin-β1 or anti-integrin-β3 antibodies. On Ti, cell adhesion was only slightly affected after a pre-treatment with anti-integrin-β3 antibodies. Scanning electron microscopy showed that on both types of substrate, cellular morphology was not changed after a pre-treatment with either antibody. With quantitative PCR, it was shown for both substrates that mRNA expression of integrin-β1 was increased after a pre-treatment with either anti-integrin-β1 or anti-integrin-β3 antibodies. Furthermore, after a pre-treatment with either antibody, mRNA expression of integrin-β3 and ALP was decreased, on both types of substrate. In conclusion, osteoblast-like cells have the ability to compensate to great extent for the blocking strategy as applied here. Still, integrin-β1 and β3 seem to play different roles in attachment, proliferation, and differentiation of osteoblast-like cells, and responses on CaP-coated substrates differ to non coated Ti. Furthermore, the influence on ALP expression suggests involvement of both integrin subunits in signal transduction for cellular differentiation

Activation of Syk protein tyrosine kinase through interaction with integrin β cytoplasmic domains

AbstractSyk protein tyrosine kinase is essential for immune system development and function [1] and for the maintenance of vascular integrity [2, 3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs) [1]. Syk can also be activated by integrin adhesion receptors [4, 5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin β3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the β3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the β3 cytoplasmic tail [β3(759X)] decreased Syk binding and disrupted its physical association with integrin αIIbβ3. Furthermore, cells expressing αIIbβ3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the αIIbβ3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin β3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase

Coordinate interactions of Csk, Src, and Syk kinases with αIIbβ3 initiate integrin signaling to the cytoskeleton

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin αIIbβ3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in αIIbβ3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with αIIbβ3. However, fibrinogen binding caused Csk to dissociate from αIIbβ3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with αIIbβ3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to αIIbβ3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton

An amphitropic cAMP-binding protein in yeast mitochondria

ABSTRACT: We describe the first example of a mitochondrial protein with a covalently attached phos-phatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cereuisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified CAMP-binding protein through a phospholipase activity. Only in recent years specific fatty acids have been recog-nized to play important roles in the association of proteins with membranes. Both noncovalent and covalent interactions be-tween fatty acids and proteins have been reported. Among the latter are GTP-binding proteins (Molenaar et al., 1988)

MoSi Superconducting Nanowire Single-Photon Detectors on GaAs for On-Chip Integration

We report on MoSi-based superconducting nanowire single-photon detectors on a gallium arsenide substrate. MoSi deposited on a passivated GaAs surface has the same critical temperature as MoSi deposited on silicon. The critical temperature decreases slightly on depositing MoSi directly on the native oxide of GaAs. Hence, MoSi works well as a thin-film superconductor on GaAs. We propose that the amorphous structure of MoSi ensures compatibility with the GaAs matrix. Superconducting nanowire single-photon detectors (SNSPDs) are fabricated with MoSi on GaAs using a meander-wire design. The SNSPD metrics are very similar to those of devices fabricated with the same procedure on a silicon substrate. We observe a plateau in the response-versus-bias curve signalling a saturated internal quantum efficiency. The plateau remains even at an elevated temperature, 2.2 K, at a wavelength of 980 nm. We achieve a timing jitter of 50 ps and a recovery time of 29 ns. These results point to the promise of integrating MoSi SNSPDs with GaAs photonic circuits