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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]

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.

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.

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]