Thomas R. Coleman

Myt1: A Membrane-Associated Inhibitory Kinase That Phosphorylates Cdc2 on Both Threonine-14 and Tyrosine-15

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phospho-rylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggested that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control.

The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts

We have cloned a Xenopus Cdc6 homolog (Xcdc6) and characterized its role in DNA replication with Xenopus egg extracts. Immunodepletion of Xcdc6 abolishes chromosomal replication but not elongation on single-stranded DNA templates. Xcdc6 binds to chromatin at the beginning of interphase but disappears from chromatin upon initiation of replication. Immunodepletion studies indicate that binding of Xcdc6 to chromatin requires Xorc2, a component of the origin recognition complex. Moreover, Xmcm3 cannot bind to chromatin lacking Xcdc6, suggesting that Xorc2, Xcdc6, and Xmcm3 associate with the DNA sequentially. In postreplicative nuclei, Xcdc6 is associated with the nuclear envelope. These studies indicate that Xcdc6, is essential for initiation of replication in vertebrates and that interaction with the nuclear envelope may regulate its function.

Cdc2 regulatory factors.

A growing family of kinases and phosphatases controls the activity of the cyclin-dependent kinase cdc2. The past year has seen the identification of the cdk activating kinase as well as considerable elucidation of the cdc25/wee1 regulatory pathways. Both cdc25 and wee1 appear to be regulated by upstream kinase/phosphatase networks. In addition, it is likely that other regulatory mechanisms cooperate with the wee1/cdc25 phosphorylation systems to control the action of cdc2. Together, these elaborate checks and balances ensure that cdc2 triggers mitosis at the appropriate time.

Two distinct mechanisms for negative regulation of the Wee1 protein kinase.

The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis‐specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2‐specific tyrosine kinase. This phosphorylation occurs in the N‐terminal region of the protein that lies outside the C‐terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1‐inhibitory kinase and a Wee1‐stimulatory phosphatase, which controls the phosphorylation of the N‐terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C‐terminal domain by the Nim1 protein and the other involving phosphorylation of its N‐terminal region by a different kinase.

Negative regulation of the weel protein kinase by direct action of the nim1/cdr1 mitotic inducer

The wee1 protein kinase suppresses the entry into mitosis by mediating the inhibitory tyrosine phosphorylation of p34cdc2. Genetic studies have suggested that the nim1 protein kinase (also known as cdr1) acts as a positive regulator of mitosis by down-regulating the wee1 pathway in yeast cells. We have overexpressed the nim1 protein in both bacteria and insect cells. The recombinant nim1 protein autophosphorylates on both tyrosine and serine residues and can phosphorylate the isolated wee1 protein directly in a cell-free system. The nim1-catalyzed phosphorylation of the wee1 protein occurs in its C-terminal region and leads to a substantial drop in its activity as a cdc2-specific tyrosine kinase. This nim1-dependent inhibition of the wee1 protein kinase can be reversed readily in vitro by treatment with a protein phosphatase. These experiments provide direct biochemical evidence that the wee1 protein is subject to negative regulation by phosphorylation and indicate that the nim1 protein acts as an inhibitory, wee1-specific kinase.

Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly

We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.