The adaptor AP2 is required for initiation of clathrin-mediated endocytosis. Kadlecova et al. delineate the functional hierarchy of AP2 interactions with phosphatidylinositol lipids and cargo and their relationship to distinct steps in clathrin-coated pit nucleation and maturation in living cells.

Clathrin-mediated endocytosis (CME) regulates the uptake of cell-surface receptors as well as their downstream signaling activities. We recently reported that signaling can reciprocally regulate CME in cancer cells and that this crosstalk can contribute to cancer progression. To further explore the nature and extent of the crosstalk between signaling and CME in cancer cell biology, we analyzed a panel of oncogenic signaling kinase inhibitors for their effects on CME across a panel of normal and cancerous cells. Inhibition of several kinases selectively affected CME in cancer cells, including inhibition of ERK1/2, which selectively inhibited CME by decreasing the rate of clathrin-coated pit (CCP) initiation. We identified an ERK1/2 substrate, the FCH/F-BAR and SH3 domain-containing protein FCHSD2, as being essential for the ERK1/2-dependent effects on CME and CCP initiation. Our data suggest that ERK1/2 phosphorylation activates FCHSD2 and regulates EGF receptor (EGFR) endocytic trafficking as well as downstream signaling activities. Loss of FCHSD2 activity in nonsmall cell lung cancer (NSCLC) cells leads to increased cell-surface expression and altered signaling downstream of EGFR, resulting in enhanced cell proliferation and migration. The expression level of FCHSD2 is positively correlated with higher NSCLC patient survival rates, suggesting that FCHSD2 can negatively affect cancer progression. These findings provide insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.

Clathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamin’s GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamin’s stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamin’s basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several “hits” that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin’s basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for more potent and chemically desirable dynamin inhibitors.

19Clathrin-mediated endocytosis is the major pathway by which cells internalize 20 materials from the external environment. Dynamin, a large multidomain GTPase, is a 21 key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated 22 clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of 23 clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors 24 of dynamin's GTPase activity, such as Dynasore and Dyngo-4a, are currently available, 25 although their specificity has been brought into question. Previous screens for these 26 inhibitors measured dynamin's stimulated GTPase activity due to lack of sufficient 27 sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We 28 report a highly sensitive fluorescence-based assay capable of detecting dynamin's 29 basal GTPase activity under conditions compatible with high throughput screening. 30Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and 31 identified several "hits" that inhibit the basal GTPase activity of dynamin-1. Subsequent 32 dose-response curves were used to validate the activity of these compounds. 33Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin's basal 34GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay 35 provides the basis for a more extensive search for robust dynamin inhibitors. 36 37 peer-reviewed)

14Clathrin-mediated endocytosis (CME) regulates the uptake of cell surface receptors, as well as 15 their downstream signaling activities. We recently reported that signaling reciprocally regulates 16 CME in cancer cells and that the crosstalk can contribute to cancer progression. To further explore 17 the nature and extent of the crosstalk between signaling and CME in cancer cell biology, we 18 analyzed a panel of oncogenic signaling kinase inhibitors for their effects on CME. Inhibition of 19 several kinases selectively affected CME function in cancer cells. Among these, ERK1/2 inhibition 20 selectively inhibited CME in cancer cells by decreasing the rate of CCP initiation. We identified 21 an ERK1/2 substrate, the FCH/F-BAR and SH3 domain-containing protein, FCHSD2, as being 22 30 31 Keywords: Epidermal growth factor receptor (EGFR); signal transduction; non-small cell lung 32 cancer (NSCLC); Nervous Wreck (Nwk) 33 34 35 3 Significance 36Clathrin-mediated endocytosis (CME) determines the internalization of receptors and their 37 downstream signaling. We discovered that CME is differentially regulated by specific signaling 38 kinases in cancer cells. In particular, ERK1/2-mediated phosphorylation of the FCH/F-BAR and 39 double SH3 domains-containing protein 2 (FCHSD2) regulates CME, and the trafficking and 40 signaling activities of EGF receptors. This reciprocal interaction negatively regulates cancer 41 proliferation and migration. The expression level of FCHSD2 is positively correlated with higher 42 cancer patient survival rates. This study identifies signaling pathways that impinge on the 43 endocytic machinery and reveals a molecular nexus for crosstalk between intracellular signaling 44 and CME. Cancer cells specifically adapt this crosstalk as a determinant for tumor progression, 45 which has implications for novel therapeutics against cancers. 46 47 48