MAP kinase pathways: molecular plug-and-play chips for the cell

Mitogen-activated protein kinase (MAPK) pathways transduce a large variety of external signals in mammals, unicellular eukaryotes, and plants. In recent years, plant MAPK pathways have attracted increasing interest resulting in the isolation of a large number of different components. Studies on the function of these components have revealed that MAPKs play important roles in the response to a broad variety of stresses, but also in the signaling of plant hormones and the cell cycle. Besides giving an update on recent results, the success and logic of MAPK-based signal transduction cascades is discussed.     

Isolation and characterization of a tobacco cDNA clone encoding a putative MAP kinase

We have isolated and sequenced a MAP (mitogen-activated protein) kinase-type cDNA from a tobacco (Nicotiana tabacum L.) cell suspension cDNA library by screening with a PCR fragment amplified from the same library with oligonucleotide primers corresponding to two sequences conserved in yeast and animal MAP kinases. The tobacco sequence, ntf3, shows 45–54% identity to various members of the MAP kinase family at the protein level. Northern experiments showed that ntf3 is expressed in all tobacco tissues tested, including pollen isolated at different developmental stages. Southern analysis indicated that, as in other organisms, there is a family of MAP kinase genes in tobacco. In complementary tests, ntf3 could not substitute the yeast MAP kinase genes fus3 and kss1.     

Recovery and characterization of amino acid analog‐resistant carrot and tobacco suspension‐cultured cells after cryostorage for over 10 years

Several Daucus carota (carrot) and Nicotiana tabacum (tobacco) cell lines that had been selected as resistant to Pro, Trp, Lys and Met analogs were thawed after over 10 years of cryostorage in liquid nitrogen. Some of these cell lines had been cloned. Viable cells were recovered in all cases, and growing lines were recovered from the carrot lines when placed either on feeder plates or directly into liquid medium. Some tobacco cultures were recovered only in liquid medium, but two Trp analog‐resistant cloned lines could not be recovered after several attempts. Generally the cryopreserved lines retained the original resistance and corresponding free amino acid levels while the same lines maintained as callus with monthly transfers for the same period often lost the resistance. Our study shows that carrot and some, but not all, tobacco cultured cell lines can be cryopreserved for over 10 years and still be recovered with their original characteristics.     

Stability of salt tolerance at the cell level after regeneration of plants from a salt tolerant tobacco cell line

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants.
The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.     

Culture-induced changes in osmolality of tobacco cell suspensions using four exogenous sugars

Suspension cultures of tobacco cells were grown in B5 media supplemented with sucrose, glucose, mannitol and sorbitol as exogenous sugars to examine culture-induced changes in the osmolality of the medium. Osmolality decreases were greatest in sucrose and glucose media during the 14 days in culture, and in glucose media were essentially linear, presumably reflecting the use of this sugar as a food source. Osmolality decreases occurred during the first week of culture in mannitol- and sorbitol-supplemented media, but later stabilized. Fresh weight of cultured cells in sucrose- and glucose-supplemented media increased by <200% during 14 days in culture, whereas cultured cells in mannitol- and sorbitol-supplemented media increased by only 39 and 48%, respectively. Cells transferred to the original liquid medium (B5 medium with 3% sucrose and 3% glucose) grew vigorously if they had been cultured in sucrose- and glucose-supplemented media; however, cells grown in mannitol- and sorbitol-supplemented media needed to be subcultured several times to recover their normal growth rate. By subculturing cells into increasingly higher conditions of sugars, cells tolerant to 560 mOsmol kg-1 H2O were obtained. The high osmolality-adapted cells increased by 140% in fresh weight in 8% glucose-supplemented medium. Glucose was best suited for producing the high osmolality required because sucrose concentrations at 10% sucrose and above resulted in cell death. To limit the decrease of osmolality in these suspension cultures requires changing the medium every 3 days to maintain osmolality above the 530 mOsmol kg-1 H2O needed to co-culture these as feeder cells with gametic and zygotic cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Stressing the role of MAP kinases in mitogenic stimulation

In yeast and animal cells, distinct subfamilies of mitogen-activated protein kinases (MAPKs) have evolved for transmitting different types of signals, such as the extracellular signal-regulated kinase (ERK) for mitogenic stimuli and differentiation, p38 and JUN kinase (JNK) for stress factors. Based on sequence analysis, the presently known plant MAPKs are most similar to ERKs, even though compelling evidence implies a role in various forms of biotic and abiotic stress responses. However, knowledge of their involvement in controlling proliferation is just emerging. A subgroup of the plant MAPKs, containing the alfalfa MMK3 and tobacco NTF6, are only active in mitotic cells and their localisation to the cell plate suggests a role in cytokinesis. An upstream regulator of MAPKs, the tobacco NPK1, appears to be also activated during mitosis. NPK1 might be associated and regulated by a microtubule motor protein. The localisation of NPK1 to the cell plate and its mitosis-specific activation suggest that together with NTF6 it could constitute a mitotic MAPK signalling module in tobacco. NPK1 appears to have a second role in repression of auxin-induced gene expression. MAPKs might also be involved in signalling within the meristems as suggested by the recruitement of a small G-protein to the CLAVATA 1 receptor-like protein kinase upon activation. In animal and yeast cells some of the small G-proteins relay signals from receptors to MAPK pathways.     

Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism

In response to extracellular stimuli, mitogen-activated protein kinase (MAPK, also known as ERK), which localizes to the cytoplasm in quiescent cells, translocates to the nucleus and then relocalizes to the cytoplasm again. The relocalization of nuclear MAPK to the cytoplasm was not inhibited by cycloheximide, confirming that the relocalization is achieved by nuclear export, but not synthesis, of MAPK. The nuclear export of MAPK was inhibited by leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent transport. We have then shown that MAP kinase kinase (MAPKK, also known as MEK), which mostly localizes to the cytoplasm because of its having NES, is able to shuttle between the cytoplasm and the nucleus constantly. MAPK, when injected into the nucleus, was rapidly exported from the nucleus by coinjected wild-type MAPKK, but not by the NES-disrupted MAPKK. In addition, injection of the fragment corresponding to the MAPK-binding site of MAPKK into the nucleus, which would disrupt the binding of MAPK to MAPKK in the nucleus, significantly inhibited the nuclear export of endogenous MAPK. Taken together, these results suggest that the relocalization of nuclear MAPK to the cytoplasm involves a MAPKK-dependent, active transport mechanism.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

Short-term aluminium uptake by tobacco cells: Growth dependence and evidence for internalization in a discrete peripheral region

Short-term uptake and initial localization of aluminium (Al) were investigated in cultured cells of Nicotiana tabacum L. cv. BY-2. Graphite furnace atomic absorption spectrometry and an in vivo Al-sensitive fluorometric assay, employing morin, yielded similar results in all experiments. Aluminium uptake was critically dependent on cell growth. As opposed to negligible uptake in stationary-phase cells, Al uptake (20 μ M AlCl₃, pH 4.5, 23°C) by actively growing cells was detectable within 5 min, with an initial rate of 16 nmol Al (10⁶ cells)⁻¹ h⁻¹. Increased CaCl₂ levels (up to 20 m M ), low temperature (4°C), and pre-chelation of Al to citrate greatly reduced Al uptake (by 75–90%). A pH-associated permeabilization of cells at pH 4.5, as monitored by trypan blue, was observed in some growing cells. Although permeability to trypan blue was not a requirement for Al uptake, enhanced membrane permeability at pH 4.5, relative to pH 5.6, may contribute to Al uptake. Aluminium was observed to localize mainly in a pronounced and discrete fluorescent zone at the cell periphery (2–30 μm wide), presumably in the cortical cytosol and/or the adjoining plasma membrane section, although the possibility cannot be excluded that some Al resided in the cell wall apposing this discrete region. However, as judged by the Al-morin assay, there were no detectable Al levels in the remaining, larger portion of the cell wall. The potential of the Al-morin method in Al toxicity studies is illustrated.     

The regulation of enzyme activities of the nicotine pathway in tobacco

The activities of the three enzymes of the route ornithine to the N-methyl-Δ¹-pyrrolinium salt and of the eight enzymes of the route quinolinic acid to nicotinic acid (pyridine nucleotide cycle) were determined in the roots of different Nicotiana tabacum L. cultivars and for comparison in tomato ( Lysopersicon esculentum L. cv. Vollendung) roots. Enzyme activities were further followed in different plant organs, dedifferentiated tissues, root organ cultures and in the roots after decapitation of tobacco plants. The data are in accord with the concept that the two routes are predominantly regulated by the enzymes putrescine methyltransferase (EC 2.1.1.53) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19), respectively. Further regulation involves the enzymes NAD⁺-pyrophosphatase (EC 3.6.1.22) and nicotinic acid mononucleotide glycohydrolase; these findings confirm the suggestion that the transformation to nicotinic acid is performed along two routes: either via the synthesis and degradation of NAD⁺ or by a direct step through the action of a specific glycohydrolase.     

Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells

The molecular links between cell cycle control and the regulation of programmed cell death are largely unknown in plants. Here we studied the relationship between the cell cycle and elicitor-induced cell death using synchronized tobacco BY-2 cells. Flow cytometry and fluorescence microscopy of nuclear DNA, and RNA gel-blot analyses of cell cycle-related genes revealed that the proteinaceous elicitor cryptogein induced cell cycle arrest at the G1 or G2 phase before the induction of cell death. Furthermore, the patterns of cell death induction and defence-related genes were different in different phases of the cell cycle. Constitutive treatment with cryptogein induced cell cycle arrest and cell death at the G1 or G2 phase. With transient treatment for 2 h, cell cycle arrest and cell death were only induced by treatment with the elicitor during the S or G1 phase. By contrast, the elicitor-induced production of reactive oxygen species was observed during all phases of the cell cycle. These results indicate that although recognition of the elicitor signal is cell cycle-independent, the induction of cell cycle arrest and cell death depends on the phase of the cell cycle.     

A heat-activated MAP kinase (HAMK) as a mediator of heat shock response in tobacco cells

A heat-activated MAP kinase (HAMK), immunologically related to the extracellular signal-regulated kinase (ERK) super-family of protein kinases, has been identified in BY2 cells of tobacco. The activation of HAMK at 37 degrees C was transient and detected within 2 min and reached a maximum level within 5 min. Ca(2+) chelators and channel blockers, and the known inhibitors of MEK, a MAP kinase kinase, prevented the heat activation of HAMK. This suggests that HAMK activation is part of a heat-triggered MAP kinase cascade that requires Ca(2+) influx. The heat shock protein HSP70 accumulated at 37 degrees C, but not when HAMK activation was prevented with the inhibitors of MEK or with Ca(2+) chelators or channel blockers. As previously shown for heat activation of HAMK, heat-induced accumulation of HSP70 requires membrane fluidization and reorganization of cytoskeleton. We concluded that heat-triggered HAMK cascade might play an essential role in the launching of heat shock response and hsp gene expression in tobacco cells.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Characterization of a tobacco extensin gene and regulation of its gene family in healthy plants and under various stress conditions

A genomic clone (Ext 1.4) encoding an extensin was isolated from a Nicotiana tabacum genomic library. The encoded polypeptide showed features characteristic of extensins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of one Tyr-Leu-Tyr-Lys motif suggests the possibility for one intramolecular isodityrosine cross-link whereas numerous Val-Tyr-Lys motifs may participate in intermolecular cross-links. This extensin appears to be close to an extensin already characterized in N. tabacum but very different from the Ext 1.2 extensin of N. sylvestris. The analysis of genomic DNA gel blots using probes spanning different parts of the gene showed that the Ext 1.4 gene belongs to a complex multigene family having one member originating from N. sylvestris and three members from N. tomentosiformis. The Ext 1.4 specific probe found a 1.4 kb mRNA in stems, roots, ovaries and germinating seeds of healthy plants. The Ext 1.4 gene family is strongly induced in actively dividing cell suspension cultures and after wounding of leaves or stems in conditions where root formation occurs. On the contrary, it is not induced in leaves in response to a hyperensitive reaction to a viral infection or after elicitor treatment.     

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.     

Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells

G protein-coupled receptors (GPCRs) such as angiotensin II, bradykinin and endothelin-1 (ET-1) are critically involved in the regulation of adrenal function, including aldosterone production from zona glomerulosa cells. Whereas, substantial data are available on the signaling mechanisms of ET-1 in cardiovascular tissues, such information in adrenal glomerulosa cells is lacking. Bovine adrenal glomerulosa (BAG) cells express receptors for endothelin-1 (ET-1) and their stimulation caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), extracellularly regulated signal kinases (ERK1/2), and their dependent proteins, p90 ribosomal S6 kinase (RSK-1) and CREB. ET-1 elicited these responses predominantly through activation of a G_i-linked cascade with a minor contribution from the G_q/PKC pathway. Whereas, selective inhibition of EGF-R kinase with AG1478 caused complete inhibition of EGF-induced ERK/RSK-1/CREB activation, it caused only partial reduction (30–40%) of such ET-1-induced responses. Consistent with this, inhibition of matrix metalloproteinases (MMPs) with GM6001 reduced ERK1/2 activation by ET-1, consistent with partial involvement of the MMP-dependent EGF-R activation in this cascade. Activation of ERK/RSK-1/CREB by both ET-1 and EGF was abolished by inhibition of Src, indicating its central role in ET-1 signaling in BAG cells. Moreover, the signaling characteristics of ET-1 in cultured BAG cells closely resembled those observed in clonal adrenocortical H295R cells. The ET-1-induced proliferation of BAG and H295 R cells was much smaller than that induced by Ang II or FGF. These data demonstrate that ET-1 causes ERK/RSK-1/CREB phosphorylation predominantly through activation of G_i and Src, with a minor contribution from MMP-dependent EGF-R transactivation.     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.     

Enhancement by sphingosine 1‐phosphate in vasopressin‐induced phosphoinositide hydrolysis in aortic smooth‐muscle cells: Involvement of p38 MAP kinase

We previously reported that sphingosine 1‐phosphate (S‐1‐P), a sphingomyelin metabolite, activates p44/p42 mitogen‐activated protein (MAP) kinase and p38 MAP kinase in aortic smooth‐muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C‐catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C₂‐ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S‐1‐P, which alone slightly stimulated the IPs formation, dose‐dependently amplified the AVP‐induced formation of IPs. Tumor necrosis factor‐α enhanced the AVP‐induced formation of IPs. However, S‐1‐P did not enhance the formation of IPs by NaF, a heterotrimeric GTP‐binding protein activator. Pertussis toxin inhibited the effect of S‐1‐P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S‐1‐P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S‐1‐P on the formation of IPs by AVP. SB203580 inhibited the AVP‐induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S‐1‐P. These results indicate that S‐1‐P amplifies AVP‐induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth‐muscle cells. J. Cell. Biochem. 80:46–52, 2000. © 2000 Wiley‐Liss, Inc.     

Increased N-acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells

Previous studies with tobacco (Nicotiana tabacum L.) cell suspensions indicated that elicitation of defense response (production of phytoalexins) with xylanase (1,4-β-D-xylanxylanohydrolase: EC 3.2.1.8) resulted in a dramatic acylation of phytosterols (Moreau et al. 1994). N-acylphosphatidylethanolamine (NAPE), an acylated derivative of phosphatidylethanolamine (PE), was recently demonstrated to be synthesized in vivo in plant tissues (Chapman and Moore 1993a). Here we report that acylation of PE was increased in elicitor-treated cells. NAPE levels increased 3-fold (from 1.6 to 4.8 mol% of total phospholipids) after a 2-h treatment of cell suspensions with xylanase (1 δg ml^?1). Specific activity of NAPE synthase increased in parallel with NAPE levels. Levels of NAPE and NAPE synthase activity declined during the period of 2–4 h after elicitation while levels of acylated sterolglycosides (ASG) continued to increase. Radiolabeling studies with [2^?14C]-ethanolamine confirmed that three times as much NAPE was synthesized in elicitor-treated cells compared to that in unelicited cells. Patterns of incorporation of [1-¹⁴C]-palmitic acid into membrane phospholipids in elicitor-treated cells suggested that increased acylation of lipids may be a result of changes in the acyl-coenzyme A pool. Treatment of cells with purified ethylene biosynthesis-inducing xylanase (EIX; 1 δg ml^?1 cells) resulted in increased levels of NAPE synthase activity comparable to those observed with the commercial preparations of xylanase. Boiled xylanase did not elicit an increase in the specific activity of NAPE synthase. Collectively our results demonstrate that the accumulation of NAPE in tobacco cells is attributable to increased activity of NAPE synthase. This suggests that NAPE may be specifically synthesized to play a protective role in membranes of plant cells as has been suggested for membranes of damaged animal cells.     

Protein kinase activities in the growth cycle of suspension cultured plant cells

In batch suspension cultures of Nicotiana tabacum and Datura innoxia protein kinase activity extracted from the whole cells and assayed with casein as substrate was followed over the growth cycle. In one case (N. tabacum) the activity was also determined in the nuclei preparation obtained from the suspension cultured cells. Immediately at the onset of the growth curve the protein kinase level increases strongly and reaches a maximum value at the early phase of proliferation; the enzyme level from the nuclei is slightly delayed. A comparison with protein synthesis shows that protein kinases are among the first proteins synthesized in the growth cycle. Chromatographic separation of the enzymes contributing to the total activity revealed that both in the extract of whole cells and in the nuclei two enzyme species are present. Their time course is similar to that of the total protein kinase level, although the activity corresponding to the enzyme with the higher molecular weight in the case of the whole cell extract is slightly delayed. The possible significance of protein phosphorylation in the growth cycle is discussed.