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Clathrin heavy chain is the structural component of the clathrin triskelion, but unique functions for the two distinct and highly conserved clathrin light chains (CLCa and CLCb, also known as CLTA and CLTB, respectively) have been elusive. Here, we show that following detachment and replating, CLCa is uniquely responsible for promoting efficient cell spreading and migration. Selective depletion of CLCa, but not of CLCb, reduced the initial phase of isotropic spreading of HeLa, H1299 and HEK293 cells by 60-80% compared to siRNA controls, and wound closure and motility by ~50%. Surface levels of ß1-integrins were unaffected by CLCa depletion. However, CLCa was required for effective targeting of FAK (also known as PTK2) and paxillin to the adherent surface of spreading cells, for integrin-mediated activation of Src, FAK and paxillin, and for maturation of focal adhesions, but not their microtubule-based turnover. Depletion of CLCa also blocked the interaction of clathrin with the nucleation-promoting factor WAVE complex, and altered actin distribution. Furthermore, preferential recruitment of CLCa to budding protrusions was also observed. These results comprise the first identification of CLCa-specific functions, with implications for normal and neoplastic integrin-based signaling and cell migration. [ABSTRACT FROM AUTHOR]

Clathrin heavy chain is the structural component of the clathrin triskelion, but unique functions for the two distinct and highly conserved clathrin light chains (CLCa and CLCb) have been elusive. Here we show that following detachment and replating, CLCa is uniquely responsible for promoting efficient cell spreading and migration. Selective depletion of CLCa, but not of CLCb, reduced the initial phase of isotropic spreading of HeLa, H1299 and HEK293 cells by 60-80% compared to siRNA controls, and wound closure and motility by ≈50%. Surface levels of β1-integrins were unaffected by CLCa depletion. However, CLCa was required for effective targeting of FAK and paxillin to the adherent surface of spreading cells, for integrin-mediated activation of Src, FAK and paxillin, and for maturation of focal adhesions but not their microtubule-based turnover. Depletion of CLCa also blocked interaction of clathrin with the nucleation promoting factor WAVE, and altered actin distribution. Further, preferential recruitment of CLCa to budding protrusions was also observed. These results comprise the first identification of CLCa-specific functions, with implications for normal and neoplastic integrin-based signaling and cell migration. [ABSTRACT FROM AUTHOR]

The functional significance of the widespread down-regulation of Rap1 GTPase-activating protein (Rap1GAP), a negative regulator of Rap activity, in human tumors is unknown. Here we show that human colon cancer cells depleted of Rap1GAP are endowed with more aggressive migratory and invasive properties. Silencing Rap1GAP enhanced the migration of confluent and single cells. In the latter, migration distance, velocity, and directionality were increased. Enhanced migration was a consequence of increased endogenous Rap activity as silencing Rap expression selectively abolished the migration of Rap1GAP-depleted cells. ROCK-mediated cell contractility was suppressed in Rap1GAP-depleted cells, which exhibited a spindle-shaped morphology and abundant membrane protrusions. Tumor cells can switch between Rho/ROCK-mediated contractility-based migration and Rac1-mediated mesenchymal motility. Strikingly, the migration of Rap1GAP-depleted, but not control cells required Rac1 activity, suggesting that loss of Rap1GAP alters migratory mechanisms. Inhibition of Rac1 activity restored membrane blebbing and increased ROCK activity in Rap1GAP- depleted cells, suggesting that Rac1 contributes to the suppression of contractility. Collectively, these findings identify Rap1GAP as a critical regulator of aggressive tumor cell behavior and suggest that the level of Rap1GAP expression influences the migratory mechanisms that are operative in tumor cells. [ABSTRACT FROM AUTHOR]

Beyond regulating Rap activity, little is known regarding the regulation and function of the Rap GTPase-activating protein Rap1GAP. Tuberin and E6TP1 protein levels are tightly regulated through ubiquitin-mediated proteolysis. A role for these RapGAPs, along with SPA-1, as tumor suppressors has been demonstrated. Whether RaplGAP performs a similar role was investigated. We now report that Rap1GAP protein levels are dynamically regulated in thyroid-stimulating hormone (TSH)-dependent thyroid cells. Upon TSH withdrawal, Rap1GAP undergoes a net increase in phosphorylation followed by proteasome-mediated degradation. Sequence analysis identified two putative destruction boxes in the Rap1GAP C-terminal domain. Glycogen synthase kinase 3β (GSK3β) phosphorylated Rap1GAP immunoprecipitated from thyroid cells, and GSK3β inhibitors prevented phosphorylation and degradation of endogenous Rap1GAP. Co-expression of GSK3β and Rap1GAP in human embryonic kidney 293 cells stimulated proteasome-dependent Rap1GAP turnover. Mutational analysis established a role for serine 525 in the regulation of Rap1GAP stability. Overexpression of Rap1GAP in thyroid cells impaired TSH/cAMPstimulated p70S6 kinase activity and cell proliferation. These data are the first to show that Rap1GAP protein levels are tightly regulated and are the first to support a role for Rap1GAP as a tumor suppressor. [ABSTRACT FROM AUTHOR]

Examines the role of cyclic adenylic acid (cAMP) as a survival factor and the signaling pathways through which cAMP affords protection. Role of P13K in cell survival; Information on wistar rat thyroid cells; Background on apoptosis.

In addition to protein kinase A (PKA), cAMP regulates the activity of cAMP-gated channels and Rap1-specific guanine nucleotide exchange factors. We tested the hypothesis that the targets of cAMP might also include regulators of the Ras protooncogene. In rat thyroid cells, thyrotropin (TSH) stimulates proliferation through a cAMP-mediated pathway that requires Ras activity. Interference with Ras impairs DNA synthesis stimulated by TSH as well as cAMP elevating agents and analogs, demonstrating that the requirement for Ras lies downstream of cAMP. Although cAMP stimulates proliferation, microinjection of the purified PKA catalytic subunit failed to do so, suggesting that factors in addition to PKA are required for cAMP-stimulated cell cycle progression. When added to thyroid cells expressing human Ha-Ras, TSH rapidly and markedly increased the proportion of GTP-bound Ras. Ras activity was increased within 1 min of TSH addition, maximal at 5–15 min, and declined to basal levels 30–60 min after hormone treatment. Cyclic AMP elevating agents elicited similar effects on Ras, indicating that TSH activates Ras through a cAMP-mediated pathway. Although cAMP-mediated, Ras activation by TSH and cAMP was independent of PKA activity. Moreover, cAMP-stimulated Ras activation was not impaired by tyrosine kinase inhibitors. These results indicate that cAMP activates targets in addition to PKA in thyroid cells, and that these targets may include regulators of Ras. The ability of cAMP elevating agents to activate Ras in addition to PKA may explain the inability of the PKA catalytic subunit to stimulate DNA synthesis in thyroid cells. Oncogene (2000) 19, 3609–3615 [ABSTRACT FROM AUTHOR]

Studies the activation of extracellular signal-regulated protein kinase (ERK) by calcium store depletion in rat liver epithelial cells. Effect of thapsigarin on activation of extracellular signal-regulated protein kinase in rat liver epithelial cells; Measurement of cytosolic free calcium.

Presents a study that determined if extracellular signal-regulated protein kinase can be activated through depletion of stored calcium in the epithelial cells of liver rat. Mechanism of calcium release from epithelial cells; Methodology; Results; Discussion.

Examines the differential translocation of protein kinase C isozymes by phorbol esters, epidermal growth factor (EGF), and angiotensin II (ANG II) in rat liver WB cells. Distribution of protein kinase C (PKC)-alpha in WB cells; Role of different PKC isoforms in signal transduction pathways.

Provides information on a study examining the spatial distribution of PCk-..., -... and -... in rat liver epithelial cells. Methodology and materials used to conduct the study; Results of the study.

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, ß-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-L-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression [ABSTRACT FROM AUTHOR]

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors. [ABSTRACT FROM AUTHOR]

Abstract: TULA, a recently identified UBA- and SH3-containing protein, has previously been shown to regulate cell signaling through protein tyrosine kinases. In order to search for novel functions of TULA, we identified, using mass spectrometry, proteins associated with TULA. ABCE-1 also known as RLI and HP68, a host factor of HIV-1 assembly, was found among TULA-associated proteins in these experiments. Considering an important role of ABCE-1 in HIV-1 assembly, we were compelled to analyze the effect of TULA on HIV-1 biogenesis. Our study provides evidence that TULA proteins substantially inhibit production of both sub-genomic and full-length HIV-1 viral particles and that the effect of TULA is dependent on UBA domain-mediated interactions. The primary role of ABCE-1 in the effect of TULA appears to be the recruitment of TULA to the sites of HIV-1 assembly where TULA interferes with the late steps of the HIV-1 life cycle, most likely by disrupting essential ubiquitylation-dependent events that remain to be identified. [Copyright &y& Elsevier]

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential. [ABSTRACT FROM AUTHOR]