Characterization of the GlnK protein of Escherichia coli.
The GlnK and PII signal transduction proteins are paralogues that play distinct roles in nitrogen regulation. Although cells lacking GlnK appear to have normal nitrogen regulation, in the absence of PII, the GlnK protein controls nitrogen assimilation by regulating the activities of the PII receptors glutamine synthetase adenylyltransferase (ATase) and the kinase/phosphatase nitrogen regulator II (NRII or NtrB), which controls transcription from nitrogen-regulated promoters. Here, the wild-type GlnK protein and two mutant forms of GlnK were purified, and their activities were compared with those of PII using purified components. GlnK and PII were observed to have unique properties. Both PII and GlnK were potent activators of the phosphatase activity of NRII, although PII was slightly more active. In contrast, PII was approximately 40-fold more potent than GlnK in the activation of the adenylylation of glutamine synthetase by ATase. While both GlnK and PII were readily uridylylated by the uridylyltransferase activity of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), only PII approximately UMP was effectively deuridylylated by the UR activity of the UTase/UR. Finally, there were subtle differences in the regulation of GlnK activity by the small molecule effector 2-ketoglutarate compared with the regulation of PII activity by this effector. Altogether, these results suggest that GlnK is unlikely to play a significant role in the regulation of ATase in wild-type cells, and that the main role of GlnK may be to contribute to the regulation of NRII and perhaps additional, unknown receptors in nitrogen-starved cells. Also, the slow deuridylylation of GlnK approximately UMP by the UTase/UR suggests that rapid interconversion of GlnK between uridylylated and unmodified forms is not necessary for GlnK function. One mutant form of GlnK, containing the alteration R47W, was observed to lack specifically the ability to activate the NRII phosphatase in vitro; it was able to be uridylylated by the UTase/UR and to activate the adenylylation activity of ATase. Another mutant form of GlnK, containing the Y51N alteration at the site of uridylylation, was not uridylylated by the UTase/UR and was defective in the activation of both the NRII phosphatase activity and the ATase adenylylation activity.
Atkinson
M R
MR
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA.
Ninfa
A J
AJ
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D009584:Nitrogen / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; C113488:GlnK protein, Azorhizobium caulinodans; D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; D010749:Phosphoprotein Phosphatases; D009584:Nitrogen