Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKA-mediated lipolysis, we have expressed constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect.     

Alcohol preference in AXB/BXA recombinant inbred mice: gender differences and gender-specific quantitative trait loci

The purpose of the present study was to characterize the C57BL/6J, A/J, and AXB/BXA Recombinant Inbred (RI) strains of mice for voluntary alcohol consumption. Quantitative Trait Locus (QTL) analysis was used to provide provisional location of QTLs for alcohol consumption. The inbred strains were screened for levels of alcohol intake (calculated as alcohol preference and absolute alcohol consumption) by receiving 4 days of forced exposure to a 10% (wt/vol) solution of alcohol, followed by 3 weeks of free choice between water and 10% alcohol. A wide and continuous distribution of values for alcohol consumption and preference was obtained in the AXB/BXA RI strains, confirming polygenic influences on alcohol-related behaviors. Significant gender differences were found for both alcohol preference [F_28,651= 2.12, p < 0.001] and absolute alcohol consumption [F_28,647= 2.57, p < 0.001]. In males, putative QTLs were mapped to chromosomes (Chrs) 2, 5, 7, 10, 11, and 16. Multiple regression analysis indicated that approximately 75% of the genetic variance in alcohol preference in males could be accounted for by three of the QTL regions. Several of the putative QTLs appeared to be male-specific (Tyr on Chr 7; D10Mit126 on Chr 10; D11Mit61 on Chr 11). In females, seven putative QTLs were mapped to Chrs 2, 4, 5, 7, 11, 16, and 19. Approximately 90% of the genetic variance in alcohol preference in females could be accounted for by four QTL regions, as determined by multiple regression. The QTL on Chr 11 near D11Mit35 appeared to be female-specific. This site was close to a female-specific QTL (Alcp2) previously mapped in C57BL/6J × DBA/2J backcrosses by Melo and coworkers (Nat Genet 13, 147, 1996). The QTLs mapped for alcohol preference in the present study must be considered suggestive at the present time, since only D2Mit74 met very strict statistical criteria for significance. However, the concordance across several studies for the loci on Chrs 2, 4, 7, 9, and 11 suggest that some common QTLs influencing alcohol preference have been identified. Confirmation of QTLs mapped in the present study is currently being conducted in a new series of recombinant congenic (RC) strains developed from reciprocal backcrosses between the A/J and C57BL/6J progenitors. The concomitant use of both RI and RC strains developed from the same progenitors should provide a powerful means of detecting, confirming, and mapping QTLs for alcohol-related traits. Received: 25 August 1998 / Accepted: 8 October 1998     

Organization of functional domains in the docking protein p130Cas

The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.     

Biomechanics of conidial dispersal in the toxic mold Stachybotrys chartarum

Conidial dispersal in Stachybotrys chartarum in response to low-velocity airflow was studied using a microflow apparatus. The maximum rate of spore release occurred during the first 5 min of airflow, followed by a dramatic reduction in dispersal that left more than 99% of the conidia attached to their conidiophores. Micromanipulation of undisturbed colonies showed that micronewton (microN) forces were needed to dislodge spore clusters from their supporting conidiophores. Calculations show that airspeeds that normally prevail in the indoor environment disturb colonies with forces that are 1000-fold lower, in the nanonewton (nN) range. Low-velocity airflow does not, therefore, cause sufficient disturbance to disperse a large proportion of the conidia of S. chartarum.     

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.