Biomedical subjects

Paul R Mueller

Publications and source records attributed to Paul R Mueller.

4 recordsLinked to original sources

PubMed2005

Measurement of Wee kinase activity.

The Wee kinases (Wee1, Wee2, and Myt1) are major regulators of mitotic entry. They function by phosphorylating Cdc2 and related Cdks on conserved tyrosine and threonine residues. This phosphorylation blocks the activity of the Cdc2 and prevents entry into mitosis. The abundance and activity of the Wee kinases are regulated during the cell cycle and development. In this chapter, we describe several procedures to measure the activity of the Wee kinases found either in crude extracts or in purified preparations. Specific protocols include the production and purification of recombinant Cdc2/Cyclin B substrate, the production of crude subcellular extract fractions, the purification of endogenous or recombinant Wee kinases, Wee kinase assays, and the Histone H1 kinase assay to measure Cdc2 activity. In addition, support protocols are provided that describe the use and production of Ni-IDA beads for the purification of Histidine-tagged proteins, and the use of the baculovirus expression system to produce recombinant proteins.

Animals↗

PubMed2004

Xenopus Cds1 is regulated by DNA-dependent protein kinase and ATR during the cell cycle checkpoint response to double-stranded DNA ends.

The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus Cds1 (XCds1). Under normal cell cycle conditions, nonactivated XCds1 constitutively associates with a Xenopus ATR complex. The association of XCds1 with this complex does not require a functional forkhead activation domain but does require a putative SH3 binding region that is found in XCds1. In response to double-stranded DNA ends, the amino terminus of XCds1 is rapidly phosphorylated in a sequential pattern. First DNA-PK phosphorylates serine 39, a site not previously recognized as important in Cds1 regulation. Xenopus ATM, ATR, and/or DNA-PK then phosphorylate three consensus serine/glutamine sites. Together, these phosphorylations have the dual function of inducing dissociation from the ATR complex and independently promoting the full activation of XCds1. Thus, the checkpoint-mediated activation of XCds1 requires phosphorylation by multiple phosphoinositide 3-kinase-related kinases, protein-protein dissociation, and autophosphorylation.

Amino Acid Sequence↗

PubMed2004

Inhibition of the cell cycle is required for convergent extension of the paraxial mesoderm during Xenopus neurulation.

Coordination of morphogenesis and cell proliferation is essential during development. In Xenopus, cell divisions are rapid and synchronous early in development but then slow and become spatially restricted during gastrulation and neurulation. One tissue that transiently stops dividing is the paraxial mesoderm, a dynamically mobile tissue that forms the somites and body musculature of the embryo. We have found that cessation of cell proliferation is required for the proper positioning and segmentation of the paraxial mesoderm as well as the complete elongation of the Xenopus embryo. Instrumental in this cell cycle arrest is Wee2, a Cdk inhibitory kinase that is expressed in the paraxial mesoderm from mid-gastrula stages onwards. Morpholino-mediated depletion of Wee2 increases the mitotic index of the paraxial mesoderm and this results in the failure of convergent extension and somitogenesis in this tissue. Similar defects are observed if the cell cycle is inappropriately advanced by other mechanisms. Thus, the low mitotic index of the paraxial mesoderm plays an essential function in the integrated cell movements and patterning of this tissue.

Animals↗

PubMed2002

Multiple Cdk1 inhibitory kinases regulate the cell cycle during development.

The Wee kinases block entry into mitosis by phosphorylating and inhibiting the activity of the mitotic cyclin-dependent kinase, Cdk1. We have found that the various Xenopus Wee kinases have unique temporal and spatial patterns of expression during development. In addition, we have isolated and characterized a new Wee1-like kinase, Xenopus Wee2. By both in vivo and in vitro tests, Xenopus Wee2 functions as a Wee1-like kinase. The previously isolated Wee1-like kinase, Xenopus Wee1, is expressed only as maternal gene product. In contrast, Xenopus Wee2 is predominantly a zygotic gene product, while the third Wee kinase, Xenopus Myt1, is both a maternal and zygotic gene product. Concurrent with the changing levels of these Cdk inhibitory kinases, the pattern of embryonic cell division becomes asynchronous and spatially restricted in the Xenopus embryo. Interestingly, once zygotic transcription begins, Xenopus Wee2 is expressed in regions of the embryo that are devoid of mitotic cells, such as the involuting mesoderm. In contrast, Xenopus Myt1 is expressed in regions of the embryo that have high levels of proliferation, such as the developing neural tissues. The existence of multiple Wee kinases may help explain how distinct patterns of cell division arise and are regulated during development.

Amino Acid Sequence↗