Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin‐dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2‐like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk‐activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2‐cdks complexes through phosphorylation of Thr161 (or its homologues).

Activation of the cyclin‐dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2‐related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild‐type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2‐T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.

The mitotic inducer p34cdc2 requires association with a cyclin and phosphorylation on Thr161 for its activity as a protein kinase. CAK, the p34cdc2 activating kinase, was previously identified as an enzyme necessary for this activating phosphorylation. We confirm here that CAK is a protein kinase and describe its purification over 13,000‐fold from Xenopus egg extracts. We further show that CAK contains a protein identical or closely related to the previously identified Xenopus MO15 gene: p40MO15 copurifies with CAK, and an antiserum to p40MO15 specifically depletes cAK activity. CAK appears to be the only protein in Xenopus egg extracts that can activate complexes of either p34cdc2 or the closely related protein kinase, p33cdk2, with either cyclin A or cyclin B. The sequence similarity between p40MO15 and p34cdc2, and the approximately 200 kDa size of CAK, suggest that p40MO15 may itself be regulated by subunit association and by protein phosphorylations.

Chinese hamster ovary cells have been engineered to inducibly over-express the p21(CIP1) cyclin-dependent kinase inhibitor, to achieve cell cycle arrest and increase cell productivity. In p21(CIP1)-arrested cells production of antibody from a stably integrated lgG4 gene, was enhanced approximately fourfold. The underlying physiological basis for enhanced productivity was investigated by measuring a range of cellular and metabolic parameters. Interestingly, the average cell volume of arrested cells was approximately fourfold greater than that of proliferating cells. This was accompanied by significant increases in mitochondrial mass, mitochondrial activity, and ribosomal protein S6 levels. Our results suggest that p21(CIP1)-induced cell cycle arrest uncouples cell growth from cell-cycle progression, and provides new insight into how improved productivity can be achieved in a cell line commonly used for large-scale production of pharmaceutical proteins.

Abstract. Soil moisture status in land surface models (LSMs) can be updated by assimilating cosmic-ray neutron intensity measured in air above the surface. This requires a fast and accurate model to calculate the neutron intensity from the profiles of soil moisture modeled by the LSM. The existing Monte Carlo N-Particle eXtended (MCNPX) model is sufficiently accurate but too slow to be practical in the context of data assimilation. Consequently an alternative and efficient model is needed which can be calibrated accurately to reproduce the calculations made by MCNPX and used to substitute for MCNPX during data assimilation. This paper describes the construction and calibration of such a model, COsmic-ray Soil Moisture Interaction Code (COS-MIC), which is simple, physically based and analytic, and which, because it runs at least 50 000 times faster than MC-NPX, is appropriate in data assimilation applications. The model includes simple descriptions of (a) degradation of the incoming high-energy neutron flux with soil depth, (b) creation of fast neutrons at each depth in the soil, and (c) scattering of the resulting fast neutrons before they reach the soil surface, all of which processes may have parameterized dependency on the chemistry and moisture content of the soil. The site-to-site variability in the parameters used in COS-MIC is explored for 42 sample sites in the COsmic-ray Soil Moisture Observing System (COSMOS), and the comparative performance of COSMIC relative to MCNPX when applied to represent interactions between cosmic-ray neutrons and moist soil is explored. At an example site in Arizona, fast-neutron counts calculated by COSMIC from the average soil moisture profile given by an independent network of point measurements in the COSMOS probe footprint are similar to the fast-neutron intensity measured by the COSMOS probe. It was demonstrated that, when used within a data assimilation framework to assimilate COS-MOS probe counts into the Noah land surface model at the Santa Rita Experimental Range field site, the calibrated COSMIC model provided an effective mechanism for translating model-calculated soil moisture profiles into aboveground fast-neutron count when applied with two radically different approaches used to remove the bias between data and model.