Biomedical subjects

Andrew Bloecher

Publications and source records attributed to Andrew Bloecher.

4 recordsLinked to original sources

PubMed2004

N-acetylation and ubiquitin-independent proteasomal degradation of p21(Cip1).

Expression of the CDK inhibitor p21(Cip1) is tightly regulated by signals that control cell division. p21 is an unstable protein that is degraded by the proteasome; however, the pathway that leads to proteasomal degradation of p21 has proven to be enigmatic. An important issue is whether proteasomal degradation of p21 occurs independently of ubiquitylation or, alternatively, whether ubiquitylation on its N terminus is crucial. We resolve this uncertainty by showing that endogenous cellular p21 is completely acetylated at its amino terminus and is therefore not a substrate for N-ubiquitylation. We further show that inactivation of essential components of the ubiquitylation machinery does not directly impact endogenous p21 degradation. Our results underscore the importance of N-acetylation in restricting N-ubiquitylation and show, in particular, that ubiquitylation of endogenous p21 either at internal lysines or on the N terminus is unlikely to control its degradation by the proteasome.

Acetylation↗

PubMed2003

Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation.

Autophosphorylation-triggered ubiquitination has been proposed to be the major pathway regulating cyclin E protein abundance: phosphorylation of cyclin E on T380 by its associated CDK allows binding to the receptor subunit, Fbw7, of the SCFFbw7 ubiquitin ligase. We have tested this model in vivo and found it to be an inadequate representation of the pathways that regulate cyclin E degradation. We show that assembly of cyclin E into cyclin E-Cdk2 complexes is required in vivo for turnover by the Fbw7 pathway; that Cdk2 activity is required for cyclin E turnover in vivo because it phosphorylates S384; that phosphorylation of T380 in vivo does not require Cdk2 and is mediated primarily by GSK3; and that two additional phosphorylation sites, T62 and S372, are also required for turnover. Thus, cyclin E turnover is controlled by multiple biological inputs and cannot be understood in terms of autophosphorylation alone.

Animals↗

PubMed2003

A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence.

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4(V831A/F833A) to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4(V831A/F833A) is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or a bni4(V831A/F833A) mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.

Cell Division↗

PubMed2002

Essential functions of Sds22p in chromosome stability and nuclear localization of PP1.

Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed to regulate one or more functions of PP1(C) during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1(C) (Glc7p). Temperature-sensitive (Ts(-)) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37 degrees C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts(-) growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts(-) growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p

Alleles↗