The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One the best-characterized enzymes that catalyzes attachment ubiquitin to is a ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought artificially target protein SCF for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds angiogenesis inhibitor ovalicin. A chimeric compound, protein-targeting molecule 1 (Protac-1),...

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation on threonine-14 and tyrosine-15 inhibits activity enzyme prevents premature initiation Although Wee1 has been identified as phosphorylates in various organisms, threonine-14-specific not isolated. A complementary DNA was cloned from Xenopus encodes Myt1, a member family discovered to phosphorylate efficiently both tyrosine-15. Myt1 membrane-associated protein contains putative transmembrane...

Cdc2, the cyclin-dependent kinase that controls mitosis, is negatively regulated by phosphorylation on its threonine-14 and tyrosine-15 residues. Cdc25, phosphatase dephosphorylates both of these residues, undergoes activation multiple kinases at mitosis. Plx1, a associates with phosphorylates amino-terminal domain was purified extensively from Xenopus egg extracts. Cloning complementary DNA revealed Plx1 related to Polo family protein kinases. Recombinant phosphorylated Cdc25 stimulated...

The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts response to DNA replication blocks or UV-damaged DNA. is also required for the cell cycle delay that induced by unreplicated In this report, we have removed homolog of ATR (Xatr) from immunodepletion. Xatr-depleted extracts, checkpoint-associated phosphorylation abolished, and strongly compromised. Xatr recombinant vitro, but not a mutant form (Xchk1-4AQ) containing nonphosphorylatable residues its four conserved...

Akiko Kumagai and William G. Dunphy Division of Biology 216-76, Howard Hughes Medical Institute, California Institute Technology, Pasadena, 91125 USA

Wee1 inactivates the Cdc2–cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of is highly regulated cell cycle. In frog egg extracts, it has been established previously that Xenopus (Xwee1) present in a hypophosphorylated, active form and undergoes down-regulation extensive phosphorylation at M-phase. We report Xwee1 also association with 14-3-3 proteins. Binding to occurs interphase, but not M-phase, requires Ser-549. A mutant (S549A) cannot bind...

TopBP1 serves as an activator of the ATR-ATRIP complex in response to presence incompletely replicated or damaged DNA. This process involves binding ATR ATR-activating domain TopBP1, which is located between BRCT domains VI and VII. displays increased Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that N-terminal region repeats I-II essential for this checkpoint-stimulated ATR-ATRIP. The also binds specifically Rad9-Hus1-Rad1 (9-1-1) extracts. occurs via...

We have analyzed the role of protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant (Xchk1) phosphorylates mitotic inducer Cdc25 vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation Ser-287 is sufficient to confer binding 14-3-3 proteins. Egg which Xchk1 has been removed immunodepletion are strongly but not totally compromised their ability undergo a cell cycle delay response presence unreplicated DNA. Xchk1-depleted...

Cdc25, the dual-specificity phosphatase that dephosphorylates Cdc2–cyclin B complex at mitosis, is highly regulated during cell cycle. In Xenopus egg extracts, Cdc25 associated with two isoforms of 14-3-3 protein. complexed primarily 14-3-3ε and to a lesser extent 14-3-3ζ. The association these proteins varies dramatically cycle: binding high interphase but virtually absent mitosis. Interaction mediated by phosphorylation ofXenopus Ser-287, which resides in consensus site. Recombinant point...

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity Xenopus laevis egg extracts. We investigated the mechanism functional importance of Cdk this interaction using both X. extracts human cells. found that also associated with Cdk-regulated manner cells was phosphorylated within conserved consensus target sequence (on S976 S1000 humans). Recombinant...

In Dictyostelium, chemotaxis to folate during growth and cAMP aggregation is controlled via cell surface receptors.To study the role of two G, proteins (Gal G,2) in these responses, we examined physiological biochemical effects null mutations caused by antisense mutagenesis gene disruptions.Disruption G,2 results an aggregationdeficient phenotype a loss receptor-mediated functions, including activation adenylate cyclase, guanylate expression GTP-mediated decrease receptor affinity for CAMP,...

Claspin is necessary for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. ATR possesses a regulatory partner called ATRIP. We have studied respective roles ATR-ATRIP and Chk1. bound well to various DNA templates extracts. single-stranded template was weakly active. By contrast, complex on both single- double-stranded regions displayed large increase kinase activity. This observation suggests that normally undergoes upon association with...

ATM (ataxia-telangiectasia mutated) is necessary for activation of Chk1 by ATR (ATM and Rad3-related) in response to double-stranded DNA breaks (DSBs) but not replication stress. TopBP1 has been identified as a direct activator ATR. We show that regulates Xenopus phosphorylating Ser-1131 thereby strongly enhancing association with egg extracts containing mutant cannot be phosphorylated on are defective the ATR-dependent phosphorylation DSBs Thus, critical ATM-dependent following production genome.

Previous results have shown that chemotaxis and the expression of several classes genes in Dictyostelium discoideum are regulated through a cell surface cAMP receptor interacting with guanine nucleotide-binding proteins (G proteins). We now describe cloning sequencing cDNAs encoding two G alpha protein subunits from Dictyostelium. The derived amino acid sequences show they 45% identical to each other mammals yeast. Both complementary multiple mRNAs differentially expressed during...