Surface plasmon resonance study of the actin-myosin sarcomeric complex and tubulin dimers

Biosensors based on the principle of surface plasmon resonance (SPR) detection were used to measure biomolecular interactions in sarcomeres and changes of the dielectric constant of tubulin samples with varying concentration. At SPR, photons of laser light efficiently excite surface plasmons propagating along a metal (gold) film. This resonance manifests itself as a sharp minimum in the reflection of the incident laser light and occurs at a characteristic angle. The dependence of the SPR angle on the dielectric permittivity of the sample medium adjacent to the gold film allows the monitoring of molecular interactions at the surface. We present results of measurements of cross-bridge attachment/detachment within intact mouse heart muscle sarcomeres and measurements on bovine tubulin molecules pertinent to cytoskeletal signal transduction models.Comment: Submitted to Journal of Modern Optics *Corresponding author: Andreas Mershin ([email protected]

Paxillin Is Tyrosine-phosphorylated by and Preferentially Associates with the Calcium-dependent Tyrosine Kinase in Rat Liver Epithelial Cells

We and others have recently cloned a non-receptor, calcium-dependent tyrosine kinase (CADTK; also known as PYK2, CAKbeta, and RAFTK) that shares both overall domain structure and 45% amino acid identity with p125(FAK). We have studied the signaling, activation, and potential function of these related enzymes in GN4 rat liver epithelial cells that express CADTK and p125(FAK) at roughly similar levels. p125(FAK) is nearly fully tyrosine-phosphorylated in resting GN4 cells. In contrast, while CADTK is not tyrosine-autophosphorylated in untreated cells, angiotensin II increases CADTK Tyr(P) by 5-10-fold. With regard to signaling, CADTK activation is correlated with stimulation of c-Jun N-terminal kinase and p70(S6K) pathways but not with the stimulation of mitogen-activated protein kinase or p90(RSK). In this report we assessed the contribution of CADTK and p125(FAK) to tyrosine phosphorylation of focal contact proteins. In adherent GN4 cells, the constitutive activity of p125(FAK) was correlated with basal paxillin, tensin, and p130(CAS) tyrosine phosphorylation. A rapid increase in the tyrosine phosphorylation of each protein was detected after treatment with angiotensin II or other agonists that stimulate CADTK; the prolonged 3-4-fold increase in paxillin tyrosine phosphorylation was the most substantial change. In the WB cell line that expresses 3-fold less CADTK than GN4 cell line agonist-dependent paxillin tyrosine phosphorylation is similarly reduced. Immunoprecipitation of CADTK from GN4 cells revealed CADTK. paxillin complexes that persisted in 500 mM NaCl but not in 0.1% SDS cell lysis buffer. The complexes were largely independent of the tyrosine phosphorylation state of either protein. Surprisingly, we did not detect p125(FAK).paxillin complexes in immunoprecipitates using either of two p125(FAK) antibodies. When CADTK and p125(FAK) were transiently overexpressed in 293(T) cells, both enzymes associated with paxillin, but the avidity of CADTK appeared to be greater. In addition, in transfected 293(T) cells, complexes between CADTK and another potential substrate, p130(CAS), were detected. In summary, in GN4 rat liver epithelial cells stimulation of CADTK was highly correlated with paxillin tyrosine phosphorylation; in addition, CADTK but not p125(FAK) was complexed to paxillin at detectable levels. This suggests that agonist-dependent cytoskeletal changes in epithelial cells might proceed, in part, by CADTK-dependent mechanisms

An SH2 Domain-dependent, Phosphotyrosine-independent Interaction between Vav1 and the Mer Receptor Tyrosine Kinase: A MECHANISM FOR LOCALIZING GUANINE NUCLEOTIDE-EXCHANGE FACTOR ACTION

Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells. Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material. Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood. By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner. Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain. Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer. Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer. Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members. This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells

Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

<p>Abstract</p> <p>Background</p> <p>Mammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs) to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis.</p> <p>Results</p> <p>ACs induced MerTK expression in MECs, resulting in elevated MerTK levels at the earliest stages of involution. Loss of MerTK resulted in AC accumulation in post-lactational MerTK-deficient mammary glands, but not in Axl and Tyro3-deficient mammary glands. Increased vascularization, fibrosis, and epithelial hyperproliferation were observed in MerTK-deficient mammary glands through at least 60 days post-weaning, due to failed efferocytosis after lactation, but did not manifest in nulliparous mice. WT host-derived macrophages failed to rescue efferocytosis in transplanted MerTK-deficient mammary epithelium.</p> <p>Conclusion</p> <p>Efferocytosis by MECs through MerTK is crucial for mammary gland homeostasis and function during the post-lactational period. Efferocytosis by MECs thus limits pathologic consequences associated with the apoptotic load following lactation.</p

Calcium-dependent increase in tyrosine kinase activity stimulated by angiotensin II.

The cellular effects of numerous hormones and neurotransmitters, including the vasoactive agents angiotensin II (AngII) and [Arg8]vasopressin, are mediated in part by protein-serine threonine kinases activated by increase of cytosolic Ca2+ concentration. In this study, we have tested the ability of Ca(2+)-mobilizing agents to activate cellular tyrosine kinases. Treatment of intact GN4 liver epithelial cells with AngII rapidly (less than or equal to 15 sec) increased tyrosine kinase activity measured either in unfractionated cell lysates or in anti-phosphotyrosine immune complexes from detergent-solubilized cells. Increased phosphorylation of the exogenous substrate poly(Glu80Tyr20) (3- to 4-fold over control) by immunoprecipitated kinases closely paralleled the time- and dose-dependence of the appearance of tyrosine phosphoproteins in intact cells. This effect of AngII was mimicked by thapsigargin, a Ca(2+)-elevating tumor promoter. The ability of AngII, but not epidermal growth factor, to increase tyrosine kinase activity was blocked in cells loaded with the Ca2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. Dephosphorylation of immunoprecipitated proteins by tyrosine phosphatase treatment was accompanied by a 60-70% loss in in vitro kinase activity, suggesting that the AngII-sensitive kinase(s) are activated by phosphorylation in intact cells. These findings demonstrate a link between two widely occurring signaling pathways, the tyrosine kinases and the Ca2+ second-messenger system, and suggest the possible involvement of Ca(2+)-activated tyrosine kinases in the endocrine actions of AngII and [Arg8]vasopressin

Adaptive Reprogramming of the Breast Cancer Kinome

Our understanding of cancer has grown considerably with recent advances in high-throughput genome and transcriptome sequencing, but techniques to comprehensively analyze protein activity are still in development. Methods to quantitatively measure the activation of signaling pathways within tumors at baseline and following therapeutic intervention will prove critical to the design of proper treatment regimens. Focusing on breast cancer, we present such a method to understand kinase signaling using multiplexed kinase inhibitor beads coupled with mass spectrometry (MIB/MS)

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor.

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC

Thyrotropin and insulin-like growth factor I regulation of tyrosine phosphorylation in FRTL-5 cells : interaction between cAMP-dependent and growth factor-dependent signal transduction

Pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiated the mitogenic response to insulin-like growth factor I (IGF-I) (Tramontano, D., Moses, A. C., Veneziani, B. M., and Ingbar, S. H. (1988) Endocrinology 122, 127-132; Takahashi, S.-I., Conti, M., and Van Wyk, J. J. (1990) Endocrinology 126, 736-745). The present study was undertaken to determine whether this synergism between TSH and IGF-I in FRTL-5 cells was correlated with changes in tyrosine phosphorylation of intracellular proteins. Tyrosine phosphorylation in intact cells was determined by gel electrophoresis and immunoblotting using monospecific anti-phosphotyrosine antibodies. Cells were preincubated for up to 24 h with TSH, dibutyryl cAMP, forskolin, or cholera toxin and then incubated for an additional 1 min in the absence or presence of IGF-I. As reported by others, IGF-I rapidly increased tyrosine phosphorylation of a 175-kDa protein as well as a less intense band of 90-100 kDa. Pretreatment for 6-12 h with either TSH or other agents that elevate intracellular cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of the 175-kDa substrate by 3-5-fold. Since TSH did not increase IGF receptor number of kinase activity, the effect of TSH is assumed to be exerted at a step distal to IGF receptor tyrosine kinase. Surprisingly, IGF-I-independent tyrosine phosphorylation was also increased by pretreatment with TSH. When intact cells were analyzed TSH produced a time- and concentration-dependent increase in tyrosine phosphorylation of a prominent 120-125-kDa substrate and less prominent 100- and 80-kDa substrates. Assays using Triton X-100-soluble extracts incubated with MgCl2, ATP, and orthovanadate demonstrated that TSH pretreatment increased tyrosine phosphorylation over that observed in untreated cells. In this cell-free assay, TSH pretreatment enhanced the phosphorylation of multiple substrates. These studies suggest that a cAMP stimulus that initiates a trophic effect can be propagated indirectly through multiple pathways including enhancement of tyrosine phosphorylation

An RCS-Like Retinal Dystrophy Phenotype in Mer Knockout Mice

PURPOSE. To determine whether mice that are homozygous for a targeted disruption of the Mer receptor tyrosine kinase gene (merkd) manifest a retinal dystrophy phenotype similar to RCS rats, which carry a mutation in the orthologous gene Mertk. METHODS. Eyes of merkdand C57BL/6 wild-type (WT) mice were examined by light and electron microscopy, whole-eye rhodopsin measurement, and Ganzfeld electroretinography (ERG). RESULTS. The merkdmice showed rapid, progressive degeneration of the photoreceptors (PRs). Features of the phenotype common to metkdmice and RCS rats included the absence or near absence of phagosomes in the retinal pigment epithelium (RPE) at the peak of outer segment (OS) disc shedding, accumulation of debris and whorls of membranes at the RPE-OS interface, transient supernormal rhodopsin content and OS lengths, the presence of OS vacuoles beginning at early ages, and a relatively slow removal of pyknotic PR nuclei. Most PRs were missing, and OS debris was removed by approximately postnatal day (P)45. Scotopic ERG responses were lower than age-matched WT responses and declined with PR loss. Photopic responses were preserved better than scotopic responses, corresponding with preferential cone preservation as judged histologically. ERG amplitudes were usually unmeasurable beyond P40, although a small-amplitude scotopic threshold response (STR) could still be elicited at P253 in some mice when only scattered PR nuclei remained. CONCLUSIONS. Ablation of Mer function in merkdmice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK