The mitogen-activated protein kinase (MAPK) cascade is a highly conserved series of three protein kinases implicated in diverse biological processes. Here we demonstrate that the cascade arrangement has unexpected consequences for the dynamics of MAPK signaling. We solved the rate equations for the cascade numerically and found that MAPK is predicted to behave like a highly cooperative enzyme, even though it was not assumed that any of the enzymes in the cascade were regulated cooperatively. Measurements of MAPK activation in Xenopus oocyte extracts confirmed this prediction. The stimulus/response curve of the MAPK was found to be as steep as that of a cooperative enzyme with a Hill coefficient of 4-5, well in excess of that of the classical allosteric protein hemoglobin. The shape of the MAPK stimulus/response curve may make the cascade particularly appropriate for mediating processes like mitogenesis, cell fate induction, and oocyte maturation, where a cell switches from one discrete state to another.Although the biological responses associated with mitogenactivated protein kinase (MAPK) signaling are highly varied, the basic structure of the MAPK cascade is well conserved (1-3). The cascade always consists of a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK), and a MAPK. MAPKKKs activate MAPKKs by phosphorylation at two conserved serine residues and MAPKKs activate MAPKs by phosphorylation at conserved threonine and tyrosine residues (Fig. 1). The cascade relays signals from the plasma membrane to targets in the cytoplasm and nucleus.A number of other membrane-to-nucleus signaling pathways, such as the Jak/Stat pathways and the cAMP/protein kinase A pathway, employ just a single protein kinase. Why does the MAPK cascade invariably use three kinases instead of one? The possibility that the three kinase arrangement has evolved to allow signal ramification or amplification is attractive but, as yet, not well supported by genetic or biochemical evidence. We have explored the possibility that the cascade arrangement has important consequences for the dynamics of MAPK signaling. Here we shall focus on the steady-state responses of enzymes at each level in the cascade to varying input stimuli. The stimulus/ response curve of a typical Michaelis-Menten enzyme is hyperbolic, and the enzyme responds in a graded fashion to increasing stimuli. An 81-fold increase in stimulus is needed to drive the enzyme from 10% to 90% maximal response (see for example, the MAPKKK curves in Fig. 2). However, some enzymes exhibit stimulus/response curves that are steeper or less steep than the Michaelis-Menten curve. Goldbeter and Koshland have termed these responses "ultrasensitivity" and "subsensitivity," respectively (11-13). An ultrasensitive enzyme requires less than an 81-fold increase in stimulus to drive it from 10% to 90% maximal response (for example, the MAPK and MAPKK curves in Fig. 2); a subsensitive enzyme requires more than an 81-fold increase.The term ultrasensitivity emphasizes the fact that the upstroke of th...

Background: The development of microarrays permits us to monitor transcriptomes on a genome-wide scale. To validate microarray measurements, quantitative-real time-reverse transcription PCR (Q-RT-PCR) is one of the most robust and commonly used approaches. The new challenge in gene quantification analysis is how to explicitly incorporate statistical estimation in such studies. In the realm of statistical analysis, the various available methods of the probe level normalization for microarray analysis may result in distinctly different target selections and variation in the scores for the correlation between microarray and Q-RT-PCR. Moreover, it remains a major challenge to identify a proper internal control for Q-RT-PCR when confirming microarray measurements.

An analytic strategy was followed to identify putative regulatory genes during the development of human hepatocellular carcinoma (HCC). This strategy employed a bioinformatics analysis that used a database search to identify genes, which are differentially expressed in human HCC and are also under cell cycle regulation. A novel cell cycle regulated gene (HURP) that is overexpressed in HCC was identified. Full-length cDNAs encoding the human and mouse HURP genes were isolated. They share 72 and 61% identity at the nucleotide level and aminoacid level, respectively. Endogenous levels of HURP mRNA were found to be tightly regulated during cell cycle progression as illustrated by its elevated expression in the G 2 /M phase of synchronized HeLa cells and in regenerating mouse liver after partial hepatectomy. Immunofluorescence studies revealed that hepatoma up-regulated protein (HURP) localizes to the spindle poles during mitosis. Overexpression of HURP in 293T cells resulted in an enhanced cell growth at low serum levels and at polyhema-based, anchorage-independent growth assay. Taken together, these results strongly suggest that HURP is a potential novel cell cycle regulator that may play a role in the carcinogenesis of human cancer cells.

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.