Linked Life Data

19840946

Source:http://linkedlifedata.com/resource/pubmed/id/19840946

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
51
pubmed:dateCreated
2009-12-16
pubmed:abstractText
The phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) metabolizing enzymes, the kinase PIKfyve and the phosphatase Sac3, constitute a single multiprotein complex organized by the PIKfyve regulator ArPIKfyve and its ability to homodimerize. We previously established that PIKfyve is activated within the triple PIKfyve-ArPIKfyve-Sac3 (PAS) core. These data assign an atypical function for the phosphatase in PtdIns(3,5)P(2) biosynthesis, thus raising the question of whether Sac3 retains its PtdIns(3,5)P(2) hydrolyzing activity within the PAS complex. Herein, we address the issue of Sac3 functionality by a combination of biochemical and morphological assays in triple-transfected COS cells using a battery of truncated or point mutants of the three proteins. We identified the Cpn60_TCP1 domain of PIKfyve as a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Neither Sac3 nor PIKfyve enzymatic activities affected the PAS complex formation or stability. Using the well established formation of aberrant cell vacuoles as a sensitive functional measure of localized PtdIns(3,5)P(2) reduction, we observed a mitigated vacuolar phenotype by kinase-deficient PIKfyve(K1831E) if its ArPIKfyve-Sac3 binding region was deleted, suggesting reduced Sac3 access to, and turnover of PtdIns(3,5)P(2). In contrast, PIKfyve(K1831E), which displays intact ArPIKfyve-Sac3 binding, triggered a more severe vacuolar phenotype if coexpressed with ArPIKfyve(WT)-Sac3(WT) but minimal defects when coexpressed with ArPIKfyve(WT) and phosphatase-deficient Sac3(D488A). These data indicate that Sac3 assembled in the PAS regulatory core complex is an active PtdIns(3,5)P(2) phosphatase. Based on these and other data, presented herein, we propose a model of domain interactions within the PAS core and their role in regulating the enzymatic activities.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1083-351X
pubmed:author
pubmed:issnType
Electronic
pubmed:day
18
pubmed:volume
284
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
35794-806
pubmed:dateRevised
2010-12-21
pubmed:meshHeading
pubmed:year
2009
pubmed:articleTitle
PIKfyve-ArPIKfyve-Sac3 core complex: contact sites and their consequence for Sac3 phosphatase activity and endocytic membrane homeostasis.
pubmed:affiliation
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural