Biomedical subjects

Qiong Lin

Publications and source records attributed to Qiong Lin.

9 recordsLinked to original sources

PubMed2006

Activated Cdc42-associated kinase 1 is a component of EGF receptor signaling complex and regulates EGF receptor degradation.

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.

Amino Acid Sequence↗

PubMed2006

Identification of a DOCK180-related guanine nucleotide exchange factor that is capable of mediating a positive feedback activation of Cdc42.

Cdc42, a member of the Rho subfamily of small GTPases, influences a wide range of activities including the establishment of cell polarity, migration, and the regulation of cell growth and differentiation. Here we describe the identification of an approximately 220-kDa protein that formed a stable complex with activated forms of Cdc42 and thereby showed characteristics of a downstream target/effector for this GTPase. However, molecular cloning of the cDNA encoding this protein (p220) revealed that it was highly related to Zizimin-1 and identical in sequence to a gene product in the data base designated DOCK11, which are members of the DOCK180 family of guanine nucleotide exchange factors (GEFs) for Cdc42 and Rac. Biochemical characterization shows that p220 is a specific GEF for Cdc42, with the GEF activity originating from its DHR2 (for DOCK homology region 2) domain. Nucleotide-depleted Cdc42 forms a stable complex with the DHR2 domain, whereas the binding of activated Cdc42 requires both the DHR2 domain and residues 66-126 within the amino-terminal portion of p220. Moreover, the full-length protein shows markedly higher GEF activity than the isolated DHR2 domain, whereas removal of the amino-terminal 126 amino acids necessary for binding-activated Cdc42 dramatically diminishes the activity. These and other results point to activated Cdc42 providing a positive feedback regulation of the GEF activity of p220. Thus, we refer to p220/DOCK11 as activated Cdc42-associated GEF, befitting its functional activity.

Amino Acid Sequence↗

PubMed2006

Magnetic double resonance in force microscopy.

Magnetic-resonance force microscopy is combined with cross-polarization and spin-decoupling NMR techniques to obtain double-resonance NMR signals of micrometer-scaled objects. The effective one-dimensional spatial resolution obtained in our experiments performed on a KPF6 single crystal sample is approximately 0.5 microm. The spectral linewidth of 900 Hz is sample limited. The described double-resonance techniques can introduce new chemical specificity to the magnetic-force sensor.

Journal Article↗

PubMed2006

Dimerization is required for SH3PX1 tyrosine phosphorylation in response to epidermal growth factor signalling and interaction with ACK2.

SH3PX1 [SNX9 (sorting nexin 9)] is a member of SNX super-family that is recognized by sharing a PX (phox homology) domain. We have previously shown that SH3PX1, phosphorylated by ACK2 (activated Cdc42-associated tyrosine kinase 2), regulates the degradation of EGF (epidermal growth factor) receptor. In mapping the tyrosine phosphorylation region, we found that the C-terminus of SH3PX1 is required for its tyrosine phosphorylation. Further analysis indicates that this region, known as the coiled-coil domain or the BAR (Bin-amphiphysin-Rvs homology) domain, is the dimerization domain of SH3PX1. Truncation of as little as 13 amino acid residues at the very C-terminus in the coiled-coil/BAR domain of SH3PX1 resulted in no dimerization, no ACK2-catalysed and EGF-stimulated tyrosine phosphorylation and no interaction with ACK2. The intracellular localization of SH3PX1 became dysfunctional upon truncation in the BAR domain. Taken together, our results indicate that the dimerization, which is mediated by the BAR domain, is essential for the intracellular function of SH3PX1.

Animals↗

PubMed2006

Measurement of epidermal growth factor receptor turnover and effects of Cdc42.

Ligand-induced degradation represents an essential component of the overall regulation of EGF receptor (EGFR)-coupled signal transduction. Following activation, EGFRs are monoubiquitinated, subsequently sorted by ubiquitin-interaction-based sorting machinery, and transported to multivesicular bodies (MVBs) and lysosomes for degradation. The Rho-family small G-protein, Cdc42, has been implicated in the regulation of EGFR degradation. Here we describe routine methods for assaying EGFR endocytosis and degradation. In addition, we have introduced procedures for determining the effects of Cdc42 and its downstream targets, in particular, ACK (Activated Cdc42-associated Kinase) and p85Cool-1 (Cloned out of library)/Pix (for Pak-interactive exchange factor), on EGFR degradation.

Animals↗

PubMed2004

Interaction of activated Cdc42-associated tyrosine kinase ACK2 with HSP90.

ACK2 (activated Cdc42-associated tyrosine kinase 2) is a specific downstream effector for Cdc42, a member of the Rho family of small G-proteins. ACK2 interacts with clathrin, an endocytic vesicle coating protein, and SH3PX1, a sorting nexin, and is involved in clathrin-mediated endocytosis. While searching for proteins that interact with ACK2, we found that HSP90 (heat-shock protein 90) binds to ACK2. Analysis of a series of truncation mutants of ACK2 has defined the regions within the kinase domain of ACK2 that are required for binding to HSP90. The binding of HSP90 to ACK2 is blocked upon treatment with geldanamycin, an HSP90-specific ATPase inhibitor, and is required for the in vivo kinase activity of ACK2 and its association with Cdc42. Overall, our data suggest a novel mechanism of regulation in which HSP90 serves as a regulatory component in an ACK2 functional complex and plays a role in sustaining its kinase activity.

Adenosine Triphosphatases↗

PubMed2004

Identification of the region in Cdc42 that confers the binding specificity to activated Cdc42-associated kinase.

The Rho family small G-protein Cdc42 has been implicated in a diversity of biological functions. Multiple downstream effectors have been identified. How Cdc42 discriminates the interaction with its multiple downstream effectors is not known. Activated Cdc42-associated tyrosine kinase (ACK) is a very specific effector of Cdc42. To delineate the Cdc42 signaling pathway mediated by ACK, we set about to identify the specific ACK-binding region in Cdc42. We utilized TC10, another member of the Rho family of G-proteins that is 66.7% identical to Cdc42, to construct TC10/Cdc42 chimeras for screening the specific ACK-binding region in Cdc42. A region between switch I and switch II has been identified as the specific ACK-binding (AB) region. The replacement of the AB region with the corresponding region in TC10 resulted in the complete loss of ACK-binding ability but did not affect the binding to WASP, suggesting that the AB region confers the binding specificity to ACK. On the other hand, replacement of the corresponding region of TC10 with the AB region enabled TC10 to acquire ACK-binding ability. Eight residues are different between the AB region and the corresponding region of TC10. The mutational analysis indicated that all eight residues contribute to the binding to ACK2. The assays for the Cdc42-mediated activation of ACK2 indicated that the AB region is essential for Cdc42 to activate ACK2 in cells. Thus, our studies have defined a specific ACK-binding region in Cdc42 and have provided a molecular basis for generating ACK binding-defective mutants of Cdc42 to delineate ACK-mediated signaling pathway.

Amino Acid Sequence↗

PubMed2003

RhoGDI is required for Cdc42-mediated cellular transformation.

BACKGROUND: Cdc42, a Rho-related small GTP binding protein, plays pivotal roles in actin cytoskeletal organization, Golgi vesicular trafficking, receptor endocytosis, and cell cycle progression. However, the target/effectors mediating these cellular activities and, in particular, those responsible for Cdc42-mediated cell growth regulation and transformation are still being determined. In this study, we set out to examine how the regulatory protein RhoGDI influences the cellular responses elicited by activated Cdc42. RESULTS: X-ray crystallographic analysis of the Cdc42-RhoGDI complex suggested that arginine 66 of Cdc42 is essential for its interaction with RhoGDI. Here we show that mutation of either arginine 66 or arginine 68 within the Switch II domain of Cdc42 completely abolished the binding of Cdc42 to RhoGDI without affecting the binding of other known regulators or target/effectors of this GTP binding protein. Introduction of the RhoGDI binding-defective mutation R66A within a constitutively active Cdc42(F28L) background was accompanied by changes in cell shape and an accumulation of Cdc42 in the Golgi when these cells were compared to those expressing Cdc42(F28L). However, the most striking change was that unlike Cdc42(F28L), which was able to induce the transformation of NIH 3T3 fibroblasts as assayed by their growth in low serum or their ability to form colonies in soft-agar, the Cdc42(F28L,R66A) mutant was transformation-defective. Likewise, the introduction of RhoGDI siRNA into Cdc42(F28L)-transfected cells inhibited their transformation. CONCLUSIONS: Taken together, the results reported here indicate that despite being a negative regulator of Cdc42 activation and GTP hydrolysis, RhoGDI plays an essential role in Cdc42-mediated cellular transformation.

Animals↗

PubMed2002

The Cdc42 target ACK2 interacts with sorting nexin 9 (SH3PX1) to regulate epidermal growth factor receptor degradation.

Activated Cdc42-associated kinase-2 (ACK2) is a non-receptor tyrosine kinase that serves as a specific effector for Cdc42, a Rho family small G-protein. Recently, we have found that ACK2 directly interacts with clathrin heavy chain through a clathrin-binding motif that is conserved in all endocytic adaptor proteins and regulates clathrin assembly, suggesting that ACK2 plays a role in clathrin-coated vesicle endocytosis (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473). Here we report the identification of another binding partner for ACK2 that has previously been implicated in endocytosis, namely the sorting nexin protein SH3PX1 (sorting nexin 9). The interaction occurs between a proline-rich domain of ACK2 and the Src homology 3 domain (SH3) of SH3PX1. Co-immunoprecipitation studies indicate that ACK2, clathrin, and SH3PX1 form a complex in cells. Epidermal growth factor (EGF) stimulated the tyrosine phosphorylation of SH3PX1, whereas co-transfection of ACK2 with SH3PX1 resulted in the constitutive phosphorylation of SH3PX1. However, co-transfection of the kinase-dead mutant ACK2(K158R) with SH3PX1 blocked EGF-induced tyrosine phosphorylation of SH3PX1, indicating that the EGF-stimulated phosphorylation of SH3PX1 is mediated by ACK2. EGF receptor levels were significantly decreased following EGF stimulation of cells co-expressing ACK2 and SH3PX1, thus highlighting a novel role for ACK2, working together with SH3PX1 to promote the degradation of the EGF receptor.

Animals↗