Biomedical subjects

Hanshuang Shao

Publications and source records attributed to Hanshuang Shao.

3 recordsLinked to original sources

PubMed2006

Spatial localization of m-calpain to the plasma membrane by phosphoinositide biphosphate binding during epidermal growth factor receptor-mediated activation.

Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP(2)) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P(2) but not PI(4)P(1) or PIP(3), releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP(2) in the plasma membrane and eliminated EGF-induced calpain activation. This PIP(2)-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP(2) in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP(2) concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-gamma hydrolyzes PIP(2) in the protruding lamellipodia.

Animals↗

PubMed2005

Core promoter binding by histone-like TAF complexes.

A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNA-contacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.

Amino Acid Motifs↗

PubMed2003

Secretion of pigment epithelium-derived factor. Mutagenic study.

Pigment epithelium-derived factor (PEDF), a neurotrophic and antiangiogenic protein, is an extracellular component of the retinal interphotoreceptor matrix which has been shown to be secreted by human fetal retinal pigment epithelial cells. It belongs to the serpin superfamily and contains the typical exposed reactive center loop. The function of this loop is still unknown. In this study we used site-directed mutagenesis of the cDNA encoding PEDF to show that (a) truncation of the C-terminal tail (Pro415-Pro418) of PEDF, (b) deletion of the Pro373-Ala380 segment that resides within the reactive center loop of the protein, and (c) alanine substitution of amino-acid residues Asn391-Thr403 located within its hydrophobic core inhibit PEDF secretion, but not its transcription, by cells transfected with the various PEDF cDNAs. On the basis of the crystal structure of PEDF, these mutations are presumed to alter the protein conformation, suggesting that conservation of the 3D structure of PEDF is essential for its secretion. In addition, we show that replacement of Gly376 and Leu377 with alanine prevents PEDF secretion. As these two residues are located within the highly exposed segment of the reactive center loop, we propose a novel function for this loop in PEDF. Our results imply that the reactive center loop, specifically Gly376 and Leu377, is involved in the interaction of PEDF with components of the quality control system in the endoplasmic reticulum, thus ensuring its efficient secretion.

Animals↗