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M W Kirschner

Publications and source records attributed to M W Kirschner.

At least 19 recordsLinked to original sources
PubMed1992

Role of phosphorylation in p34cdc2 activation: identification of an activating kinase.

Phosphorylation of p34cdc2 can both positively and negatively regulate its kinase activity. We have mapped two phosphorylation sites in Xenopus p34cdc2 to Thr-14 and Tyr-15 within the putative ATP-binding region of p34cdc2. Mutation of these sites to Ala-14 and Phe-15 has no effect on the final histone H1 kinase activity of the cyclin/p34cdc2 complex. Phosphopeptide analysis shows that there is at least one more site of phosphorylation on p34cdc2. When Thr-161 is changed to Ala, two phosphopeptide spots disappear and it is no longer possible to activate the H1 kinase activity of p34cdc2. We suggest that Thr-161 is a third site of phosphorylation, which is required for kinase activity. All three phosphorylations are induced by cyclin. None of the phosphorylations appears to be required for binding to cyclin, as indicated by the ability of the triple mutant, Ala-14, Phe-15, Ala-161, to bind cyclin. The activating phosphorylation that requires Thr- or Ser-161 occurs even in a catalytically inactive K33R mutant of p34cdc2 and hence does not appear to be the result of intramolecular autophosphorylation. We have detected an activity in Xenopus extracts required for activation of p34cdc2 and present evidence that this is a p34cdc2 activating kinase which, in a cyclin-dependent manner, probably directly phosphorylates Thr-161.

Adenosine Triphosphate↗
PubMed1992

Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau.

Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization. Tau strongly suppresses the catastrophe rate, and its ability to do so is independent of its ability to increase the elongation rate. Thus, tau generates a partially stable but still dynamic state in microtubules. This state is perturbed by phosphorylation by MAP2 kinase, which affects all three activities by lowering the affinity of tau for the microtubule lattice.

Animals↗
PubMed1991

cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2.

cdc25 controls the activity of the cyclin-p34cdc2 complex by regulating the state of tyrosine phosphorylation of p34cdc2. Drosophila cdc25 protein from two different expression systems activates inactive cyclin-p34cdc2 and induces M phase in Xenopus oocytes and egg extracts. We find that the cdc25 sequence shows weak but significant homology to a phylogenetically diverse group of protein tyrosine phosphatases. cdc25 itself is a very specific protein tyrosine phosphatase. Bacterially expressed cdc25 directly dephosphorylates bacterially expressed p34cdc2 on Tyr-15 in a minimal system devoid of eukaryotic cell components, but does not dephosphorylate other tyrosine-phosphorylated proteins at appreciable rates. In addition, mutations in the putative catalytic site abolish the in vivo activity of cdc25 and its phosphatase activity in vitro. Therefore, cdc25 is a specific protein phosphatase that dephosphorylates tyrosine and possibly threonine residues on p34cdc2 and regulates MPF activation.

Amino Acid Sequence↗
PubMed1991

Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos.

Peptide growth factors may play a role in patterning of the early embryo, particularly in the induction of mesoderm. We have explored the role of fibroblast growth factor (FGF) in early Xenopus development by expressing a dominant negative mutant form of the FGF receptor. Using a functional assay in frog oocytes, we found that a truncated form of the receptor effectively abolished wild-type receptor function. Explants from embryos expressing this dominant negative mutant failed to induce mesoderm in response to FGF. In whole embryos the mutant receptor caused specific defects in gastrulation and in posterior development, and overexpression of a wild-type receptor could rescue these developmental defects. These results demonstrate that the FGF signaling pathway plays an important role in early embryogenesis, particularly in the formation of the posterior and lateral mesoderm.

Actins↗
PubMed1991

INH, a negative regulator of MPF, is a form of protein phosphatase 2A.

MPF, a protein kinase complex consisting of cyclin and p34cdc2 subunits, promotes the G2 to M phase transition in eukaryotic cells. The pathway of activation and inactivation of MPF is not well understood, although there is strong evidence that removal of phosphate from a tyrosine residue on p34cdc2 is part of the activation process. INH was originally identified as an activity that could inhibit the posttranslational activation of a latent form of MPF, called pre-MPF, in immature (G2 phase-arrested) Xenopus oocytes. We have purified INH and demonstrated that it is a form of protein phosphatase 2A. Both INH and the catalytic subunit of protein phosphatase 2A can directly inactivate an isolated p34cdc2-cyclin complex. Both cyclin and p34cdc2 become dephosphorylated; the rate of inactivation closely parallels the removal of phosphate from a specific site on p34cdc2. We propose that INH opposes MPF activation by reversing this critical phosphorylation.

Animals↗
PubMed1991

Cyclin is degraded by the ubiquitin pathway.

Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.

Amino Acid Sequence↗
PubMed1991

Microtubule behavior in the growth cones of living neurons during axon elongation.

To understand how microtubules are generated in the growth cone, we have imaged fluorescently tagged microtubules in living frog embryonic neurons. The neurons were labeled by injecting rhodamine-labeled tubulin into the fertilized egg and explanting the neurons from the neural tube. Microtubules extend deep into the growth cone periphery and adopt three characteristic distributions: (a) dispersed and splayed throughout much of the growth cone; (b) looped and apparently contorted by compression; and (c) bundled into tight arrays. These distributions interconvert on a time scale of several minutes and these interconversions are correlated with the behavior of the growth cone. We observed microtubule growth and shrinkage in growth cones, but are unable to determine their contribution to net assembly. However, translocation of polymer form the axon appears to be a major mechanism of generating new polymer in the growth cone, while bundling of microtubules in the growth cone appears to be the critical step in generating new axon. Neurons that were about to turn spontaneously generated microtubules in the future direction of growth, suggesting that orientation of microtubules might be an important early step in neuronal pathfinding.

Animals↗
PubMed1991

Tau protein binds to microtubules through a flexible array of distributed weak sites.

Tau protein plays a role in the extension and maintenance of neuronal processes through a direct association with microtubules. To characterize the nature of this association, we have synthesized a collection of tau protein fragments and studied their binding properties. The relatively weak affinity of tau protein for microtubules (approximately 10(-7) M) is concentrated in a large region containing three or four 18 amino acid repeated binding elements. These are separated by apparently flexible but less conserved linker sequences of 13-14 amino acids that do not bind. Within the repeats, the binding energy for microtubules is delocalized and derives from a series of weak interactions contributed by small groups of amino acids. These unusual characteristics suggest tau protein can assume multiple conformations and can pivot and perhaps migrate on the surface of the microtubule. The flexible structure of the tau protein binding interaction may allow it to be easily displaced from the microtubule lattice and may have important consequences for its function.

Amino Acid Sequence↗
PubMed1990

Cyclin activation of p34cdc2.

The gradual accumulation of cyclin in the frog egg induces an abrupt and concerted activation of p34cdc2 that initiates mitosis. Activation is delayed even after the accumulation of cyclin to a critical threshold concentration. We have reproduced these unusual kinetic properties of p34cdc2 activation in vitro using bacterially expressed cyclin proteins and extracts derived from Xenopus eggs. Abrupt activation follows a lag period, the length of which is independent of the concentration of cyclin. The threshold concentration of cyclin and the length of the lag period are regulated by INH, an inhibitor of MPF activation in oocytes recently identified as a type 2A protein phosphatase. Binding to cyclin induces both tyrosine and threonine phosphorylation of the previously unphosphorylated p34cdc2, rendering it inactivated. The concerted transition into mitosis involves both a reduction in the rate of p34cdc2 phosphorylation on tyrosine and an increase in its rate of dephosphorylation.

Animals↗
PubMed1990

Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C.

The mechanism by which MPF induces nuclear lamin disassembly and nuclear envelope breakdown during mitosis was studied in a frog egg extract in which the transition from interphase to mitosis can be induced by the addition of MPF. Bacterially expressed human nuclear lamin C, assembled in vitro into filaments, showed increased phosphorylation on specific sites in the extract in response to MPF. Phosphorylation was accompanied by disassembly of the lamin filaments. We determined the sequences of the sites phosphorylated both in the presence and absence of MPF. The sequence data suggest that multiple protein kinases act on the lamins, and S6 kinase II was identified as one potentially important lamin kinase.

Amino Acid Sequence↗
PubMed1990

Phosphorylation of microtubule-associated protein tau: identification of the site for Ca2(+)-calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles.

The microtubule array in neuronal cells undergoes extensive growth, dynamics and rearrangements during neurite outgrowth. While little is known about how these changes are regulated, microtubule-associated proteins (MAPs) including tau protein are likely to perform an important role. Tau is one of the MAPs in mammalian brain. When isolated it is usually a mixture of several isoforms containing between 341 and 441 residues that arise from alternative splicing. Tau can be phosphorylated by several protein kinases. Phosphorylation at certain sites results in major structural and functional changes, as seen by changes in electrophoretic mobility, interaction with microtubules, molecular length and elasticity. Here we show that the sites of phosphorylation by four kinases (PKA, PKC, CK and CaMK) all lie in the C-terminal microtubule-binding half of tau, but only the phosphorylation by CaM kinase shows the pronounced shift in electrophoretic mobility characteristic for tau from Alzheimer neurofibrillary tangles. By using a combination of limited proteolysis, protein sequencing and protein engineering we show that a single phosphorylation site is responsible for this shift, located at Ser 405 in the C-terminal tail of the protein outside the region of internal repeats. Phosphorylation at this site not only reduces the electrophoretic mobility of tau, it also makes the protein long and stiff, as shown earlier. The site is likely to be phosphorylated in tau from Alzheimer neurofibrillary tangles.

Alzheimer Disease↗
PubMed1990

Regulation of the fibroblast growth factor receptor in early Xenopus embryos.

Recent evidence suggests that fibroblast growth factor (FGF) is a primary mesoderm inducer in Xenopus development. We have isolated a full-length cDNA clone for the Xenopus FGF receptor. Like other FGF receptors, the Xenopus homolog is a membrane-spanning protein with a split intracellular tyrosine kinase domain. The Xenopus FGF receptor mRNA is present as a maternal message whose levels are constant through early development. There is no specific regional localization of the transcript by analysis of FGF receptor mRNA levels in microdissected embryonic tissue. In isolated animal-pole blastomeres, FGF receptor mRNA declines over 16 hr in culture and this loss can be prevented by incubation with FGF or activin. Despite the presence of the FGF receptor mRNA in the oocyte, oocytes in culture do not respond to added FGF. However, injection of exogenous Xenopus FGF receptor transcripts into oocytes does generate a functional response to FGF. Our data suggest that posttranscriptional response to FGF. Our data suggest that posttranscriptional mechanisms regulate the FGF receptor in the oocyte and early embryo and further suggest that mesoderm-inducing factors influence receptor mRNA levels during the time of early tissue formation.

Amino Acid Sequence↗
PubMed1989

An antisense mRNA directs the covalent modification of the transcript encoding fibroblast growth factor in Xenopus oocytes.

The Xenopus oocyte contains three maternal basic fibroblast growth factor (bFGF) transcripts, the largest of which encodes the complete bFGF polypeptide. We show that the smallest transcript is transcribed in the opposite direction of the largest transcript and overlaps part of the coding sequence of bFGF. This antisense transcript encodes a highly conserved 25 kd polypeptide. In addition, the antisense transcript causes modification of the mRNA encoding bFGF during maturation of the oocyte, converting half of the adenine residues to inosine in the region of overlap between the sense and antisense transcripts. As this activity acts only on double-stranded RNA, the two mRNAs must be hybridized in the oocyte. We discuss a possible role for the antisense transcript in regulating the stability of the bFGF mRNA.

Amino Acid Sequence↗
PubMed1989

Dominoes and clocks: the union of two views of the cell cycle.

We review the recent advances in understanding transitions within the cell cycle. These have come from both genetic and biochemical approaches. We discuss the phylogenetic conservation of the mechanisms that induce mitosis and their implications for other transitions in the cell cycle.

Animals↗
PubMed1989

Cyclin synthesis drives the early embryonic cell cycle.

We have produced extracts of frog eggs that can perform multiple cell cycles in vitro. Destruction of the endogenous messenger RNA arrests the extracts in interphase. The addition of exogenous cyclin mRNA is sufficient to produce multiple cell cycles. The newly synthesized cyclin protein accumulates during each interphase and is degraded at the end of each mitosis.

Animals↗