Differential Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by Protein Kinase CK2 Phosphorylation

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser185 by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. mutant proteins exhibited cytokine and with the both in and in the and S185A mutant proteins whereas both the and mutant proteins also that phosphorylation the properties of the in a of whereas protein cytokine activity. by which phosphorylation the enzymatic of PGI has the of the regulation of this key Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser185 by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. mutant proteins exhibited cytokine and with the both in and in the and S185A mutant proteins whereas both the and mutant proteins also that phosphorylation the properties of the in a of whereas protein cytokine activity. by which phosphorylation the enzymatic of PGI has the of the regulation of this key Phosphoglucose isomerase autocrine motility protein kinase autocrine motility protein kinase is a multifunctional protein that plays role in both the and the of and extracellularly as a and PGI as autocrine motility factor in and a which the of and a which to and a in the or of PGI to or of in associated with PGI/AMF is in and in and is of associated with progression tumor PGI as which and by motility the autocrine to a associated with PGI/AMF as a factor as to the and the of analyses of this protein been revealed that PGI is a of a and a and that which is enzymatic activity, is in the the and and the of the of PGI in of cytokine activity, that the of the enzyme with cytokine with the that the sugar and also cytokine the regulation of the of PGI of kinase and also and kinase phosphorylated by in down-regulation of kinase phosphorylation of up-regulation of enzymatic and phosphorylation of glycolysis is the or of the of the also by phosphorylation. it was that PGI is phosphorylated at Ser185 by protein kinase CK2 although the physiological significance of phosphorylation to Thus, to the of phosphorylated we by site-directed mutagenesis, and of PGI proteins and and enzymatic and cytokine and human was in with and and at in a of and Ser-to-Ala at (S185A) to abolish phosphorylation and (S185D) and to mimic generated with the site-directed human PGI/AMF was as a of the to and and as and at and the was with the the was the and the of human PGI and the changes the and mutant PGI proteins the of human PGI phosphorylated at Ser185 the with PGI and RNA and mutant PGI the with the to the was to in and and the was was to and to a which was by RNA human CK2 and as a and as with to the and was of human PGI was with or CK2 in and of at with human PGI and with and by was by was of PGI enzymatic of and mutant PGI proteins as of was to a of and of and enzymatic was at a PGI was as a in mutant PGI enzymatic activity, with and mutant PGI in and at was to enzymatic and motility was with to the of with of was to the and of with was to the a with PGI proteins at the with with and with that the the the of the with motility of with as in and a at with or PGI the by the a of at and the a was of and PGI and mutant PGI proteins the the proteins in with a of and at with phosphorylated human PGI protein was also as with and by in mutant protein with and mutant PGI in with at with and by and with and mutant PGI proteins in of and with of proteins to and with with at and in with or PGI and of and mutant PGI proteins and at with and in of was a the and with a and by of and the of Ser185 or of PGI and was that PGI by phosphorylation of Ser185 by CK2 the that phosphorylation of enzymatic we phosphorylation of PGI is of physiological we by site-directed we substituted Ser185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation and Ser185 with which phosphorylation. in the and and mutant PGI proteins as a protein revealed that whereas the and S185A proteins exhibited a enzymatic activity, the and PGI mutant PGI in we the which proteins with a the mutant proteins the PGI protein that in the with and mutant PGI and we that PGI phosphorylation PGI PGI/AMF to is phosphorylated however, that mutant PGI proteins and also and (S185A) PGI proteins in and S185A mutant PGI to PGI activity, whereas and we the enzymatic of the and mutant PGI proteins phosphorylation or acid Thus, human CK2 was to human PGI and phosphorylated PGI was by mutant PGI protein we a was as and PGI in the of CK2 enzymatic of phosphorylated PGI was and to by at the of the enzymatic was as as that in the mutant proteins which to the that of the PGI in the phosphorylation phosphorylated and to the of the mutant of phosphorylated enzymatic of phosphorylated PGI/AMF was of that of PGI/AMF in the although it was that of the and which mimic phosphorylation. of PGI/AMF phosphorylation by PGI/AMF was phosphorylated by CK2 and was phosphorylated CK2 whereas was phosphorylated with CK2 and phosphorylation of of with we with CK2 to CK2 with PGI activity. CK2 was by by and the PGI of with of CK2 PGI enzymatic activity. with CK2 and as a CK2 in decreased to in which led to up-regulation of PGI enzymatic activity. a and and and of with of Ser185 of PGI the PGI phosphorylation and cytokine activity, we to motility motility and motility and and mutant proteins at with in the of and and in the motility the mutant proteins the motility of to the as the protein to of and to that PGI phosphorylation cytokine activity, we to and that of the Ser185 mutant PGI proteins exhibited to of the protein of and mutant PGI/AMF proteins and that the in and mutant PGI/AMF proteins to the motility of with of by at and mutant PGI/AMF proteins the of and mutant PGI/AMF in with or a of PGI/AMF and of and mutant PGI/AMF proteins at and the of was and the and mutant proteins PGI/AMF proteins at PGI/AMF proteins by as and with of and PGI we PGI phosphorylation to conformational which in the in enzymatic activity. PGI is a dimeric and is enzymatic activity. Thus, to the of the in we the of the proteins in the or of the and the of and in the of the the in S185A and proteins as a protein the of the revealed that both the and S185A protein as in and whereas the and proteins both and in PGI also a in the of the as the and whereas PGI with the the of in PGI we the with the at and by in the in to a of PGI was with the and proteins and with the and S185A proteins and that the negative charge and conformational changes that mimic phosphorylation changes that the to in a in the of enzymatic and mutant PGI/AMF the and mutant proteins a the in with the the and mutant proteins a and also a of and protein and with and S185A and with and and with and and with human PGI was or with CK2 and PGI a and CK2 in the and of and with CK2 in the and of mutant PGI with the and the by and a in and PGI of with of PGI and of Ser185 the the of Ser185 phosphorylation the of we the the of PGI as a PGI is a as a with the in and in to the enzyme and the of Ser185 is and is the of the the role of Ser185 we of human PGI to phosphorylated Ser185 human PGI the of the and and changes the of human PGI phosphorylated at the of Ser185 is to the of the dimeric of it is that of the is phosphorylation. in the of the of and to the of the of changes the of and PGI/AMF dimeric of PGI/AMF with was the and mutant PGI/AMF and enzyme is and is is that Ser185 is the of the and the of the and Ser185 of dimeric human the of Ser185 is the in the the PGI/AMF was by and is in the human PGI/AMF human PGI/AMF phosphorylated at Ser185 Ser185 is by the was the and PGI/AMF the phosphorylation of human and PGI/AMF proteins in and and of Ser185 in and was in the although was at this we that human PGI/AMF is a that is phosphorylated at Ser185 by CK2 and that phosphorylation enzymatic activity. this PGI is as it with as kinase and in that phosphorylation enzymatic activity. PGI is in a of acid has whereas acid the enzyme to the the phosphorylation of PGI has also PGI Ser185 to and Ser185 in and that PGI phosphorylation is a isomerase glycolysis and and PGI as a protein that is the and it as which is in and a which the of and a which to and a PGI is in and in and is of associated with progression has that PGI phosphorylation is associated with revealed that Ser185 phosphorylation of human PGI the of the that phosphorylation with with of the sugar is by the that phosphorylation to the phosphorylation of the protein and that phosphorylation the of the protein that in the Ser185 in human PGI/AMF to in in in and Ser185 in in Ser185 is in a of the PGI enzymatic by Ser185 phosphorylation the of Ser185 this is that Ser185 is the of the a phosphorylation changes by at the enzymatic of changes by the phosphorylation abolish the enzymatic of phosphorylated by the negative charge of the the of the also the enzymatic by the negative of the of Ser185 and the the of the in the is also that the negative charge by phosphorylation the of the at Ser185 a that by or a of at the the of PGI associated with and that the PGI that and that PGI the this that phosphorylation of PGI is known that of glycolysis with and in in and protein and the enzyme of the is in a PGI to and PGI the and metabolism in a PGI to the of that cellular and the that phosphorylation of PGI enzymatic and that this to to the decreased enzymatic of phosphorylated PGI changes in or and the mutant PGI proteins generated in analyses of changes in metabolism the of the Phosphoglucose isomerase autocrine motility protein kinase autocrine motility protein kinase is a multifunctional protein that plays role in both the and the of and extracellularly as a and PGI as autocrine motility factor in and a which the of and a which to and a in the or of PGI to or of in associated with PGI/AMF is in and in and is of associated with progression tumor PGI as which and by motility the autocrine to a associated with PGI/AMF as a factor as to the and the of analyses of this protein been revealed that PGI is a of a and a and that which is enzymatic activity, is in the the and and the of the of PGI in of cytokine activity, that the of the enzyme with cytokine with the that the sugar and also cytokine the regulation of the of PGI of kinase and also and kinase phosphorylated by in down-regulation of kinase phosphorylation of up-regulation of enzymatic and phosphorylation of glycolysis is the or of the of the also by phosphorylation. it was that PGI is phosphorylated at Ser185 by protein kinase CK2 although the physiological significance of phosphorylation to Thus, to the of phosphorylated we by site-directed mutagenesis, and of PGI proteins and and enzymatic and cytokine and human was in with and and at in a of and Ser-to-Ala at (S185A) to abolish phosphorylation and (S185D) and to mimic generated with the site-directed human PGI/AMF was as a of the to and and as and at and the was with the the was the and the of human PGI and the changes the and mutant PGI proteins the of human PGI phosphorylated at Ser185 the with PGI and RNA and mutant PGI the with the to the was to in and and the was was to and to a which was by RNA human CK2 and as a and as with to the and was of human PGI was with or CK2 in and of at with human PGI and with and by was by was of PGI enzymatic of and mutant PGI proteins as of was to a of and of and enzymatic was at a PGI was as a in mutant PGI enzymatic activity, with and mutant PGI in and at was to enzymatic and motility was with to the of with of was to the and of with was to the a with PGI proteins at the with with and with that the the the of the with motility of with as in and a at with or PGI the by the a of at and the a was of and PGI and mutant PGI proteins the the proteins in with a of and at with phosphorylated human PGI protein was also as with and by in mutant protein with and mutant PGI in with at with and by and with and mutant PGI proteins in of and with of proteins to and with with at and in with or PGI and of and mutant PGI proteins and at with and in of was a the and with a and by of and the and human was in with and and at in a of and Ser-to-Ala at (S185A) to abolish phosphorylation and (S185D) and to mimic generated with the site-directed human PGI/AMF was as a of the to and and as and at and the was with the the was the and the of human PGI and the changes the and mutant PGI proteins the of human PGI phosphorylated at Ser185 the with PGI and RNA and mutant PGI the with the to the was to in and and the was was to and to a which was by RNA human CK2 and as a and as with to the and was of human PGI was with or CK2 in and of at with human PGI and with and by was by was of PGI enzymatic of and mutant PGI proteins as of was to a of and of and enzymatic was at a PGI was as a in mutant PGI enzymatic activity, with and mutant PGI in and at was to enzymatic and motility was with to the of with of was to the and of with was to the a with PGI proteins at the with with and with that the the the of the with motility of with as in and a at with or PGI the by the a of at and the a was of and PGI and mutant PGI proteins the the proteins in with a of and at with phosphorylated human PGI protein was also as with and by in mutant protein with and mutant PGI in with at with and by and with and mutant PGI proteins in of and with of proteins to and with with at and in with or PGI and of and mutant PGI proteins and at with and in of was a the and with a and by of and the of Ser185 or of PGI and was that PGI by phosphorylation of Ser185 by CK2 the that phosphorylation of enzymatic we phosphorylation of PGI is of physiological we by site-directed we substituted Ser185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation and Ser185 with which phosphorylation. in the and and mutant PGI proteins as a protein revealed that whereas the and S185A proteins exhibited a enzymatic activity, the and PGI mutant PGI in we the which proteins with a the mutant proteins the PGI protein that in the with and mutant PGI and we that PGI phosphorylation PGI PGI/AMF to is phosphorylated however, that mutant PGI proteins and also and (S185A) PGI proteins in and S185A mutant PGI to PGI activity, whereas and we the enzymatic of the and mutant PGI proteins phosphorylation or acid Thus, human CK2 was to human PGI and phosphorylated PGI was by mutant PGI protein we a was as and PGI in the of CK2 enzymatic of phosphorylated PGI was and to by at the of the enzymatic was as as that in the mutant proteins which to the that of the PGI in the phosphorylation phosphorylated and to the of the mutant we with CK2 to CK2 with PGI activity. CK2 was by by and the PGI of with of CK2 PGI enzymatic activity. with CK2 and as a CK2 in decreased to in which led to up-regulation of PGI enzymatic activity. a and and and of with of Ser185 of PGI the PGI phosphorylation and cytokine activity, we to motility motility and motility and and mutant proteins at with in the of and and in the motility the mutant proteins the motility of to the as the protein to of and to that PGI phosphorylation cytokine activity, we to and that of the Ser185 mutant PGI proteins exhibited to of the protein of and mutant PGI/AMF proteins and that the in and mutant PGI/AMF proteins to the motility of with of by at and mutant PGI/AMF proteins the of and mutant PGI/AMF in with or a of PGI/AMF and of and mutant PGI/AMF proteins at and the of was and the and mutant proteins PGI/AMF proteins at PGI/AMF proteins by as and with of and PGI we PGI phosphorylation to conformational which in the in enzymatic activity. PGI is a dimeric and is enzymatic activity. Thus, to the of the in we the of the proteins in the or of the and the of and in the of the the in S185A and proteins as a protein the of the revealed that both the and S185A protein as in and whereas the and proteins both and in PGI also a in the of the as the and whereas PGI with the the of in PGI we the with the at and by in the in to a of PGI was with the and proteins and with the and S185A proteins and that the negative charge and conformational changes that mimic phosphorylation changes that the to in a in the of enzymatic and mutant PGI/AMF the and mutant proteins a the in with the the and mutant proteins a and also a of and protein and with and S185A and with and and with and and with human PGI was or with CK2 and PGI a and CK2 in the and of and with CK2 in the and of mutant PGI with the and the by and a in and PGI of with of PGI and of Ser185 the the of Ser185 phosphorylation the of we the the of PGI as a PGI is a as a with the in and in to the enzyme and the of Ser185 is and is the of the the role of Ser185 we of human PGI to phosphorylated Ser185 human PGI the of the and and changes the of human PGI phosphorylated at the of Ser185 is to the of the dimeric of it is that of the is phosphorylation. in the of the of and to the of the of changes the of and PGI/AMF dimeric of PGI/AMF with was the and mutant PGI/AMF and enzyme is and is is that Ser185 is the of the and the of the and Ser185 of dimeric human the of Ser185 is the in the the PGI/AMF was by and is in the human PGI/AMF human PGI/AMF phosphorylated at Ser185 Ser185 is by the was the and PGI/AMF the phosphorylation of human and PGI/AMF proteins in and and of Ser185 in and was in the although was at of Ser185 or of PGI and was that PGI by phosphorylation of Ser185 by CK2 the that phosphorylation of enzymatic we phosphorylation of PGI is of physiological we by site-directed we substituted Ser185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation and Ser185 with which phosphorylation. in the and and mutant PGI proteins as a protein revealed that whereas the and S185A proteins exhibited a enzymatic activity, the and PGI mutant PGI in we the which proteins with a the mutant proteins the PGI protein that in the with and mutant PGI and we that PGI phosphorylation PGI PGI/AMF to is phosphorylated however, that mutant PGI proteins and also and (S185A) PGI proteins in and S185A mutant PGI to PGI activity, whereas and we the enzymatic of the and mutant PGI proteins phosphorylation or acid Thus, human CK2 was to human PGI and phosphorylated PGI was by mutant PGI protein we a was as and PGI in the of CK2 enzymatic of phosphorylated PGI was and to by at the of the enzymatic was as as that in the mutant proteins which to the that of the PGI in the phosphorylation phosphorylated and to the of the mutant we with CK2 to CK2 with PGI activity. CK2 was by by and the PGI of with of Ser185 of PGI the PGI phosphorylation and cytokine activity, we to motility motility and motility and and mutant proteins at with in the of and and in the motility the mutant proteins the motility of to the as the protein to of and to that PGI phosphorylation cytokine activity, we to and that of the Ser185 mutant PGI proteins exhibited to of the protein of and PGI we PGI phosphorylation to conformational which in the in enzymatic activity. PGI is a dimeric and is enzymatic activity. Thus, to the of the in we the of the proteins in the or of the and the of and in the of the the in S185A and proteins as a protein the of the revealed that both the and S185A protein as in and whereas the and proteins both and in PGI also a in the of the as the and whereas PGI with the the of in PGI we the with the at and by in the in to a of PGI was with the and proteins and with the and S185A proteins and that the negative charge and conformational changes that mimic phosphorylation changes that the to in a in the of enzymatic activity. of PGI and of Ser185 the the of Ser185 phosphorylation the of we the the of PGI as a PGI is a as a with the in and in to the enzyme and the of Ser185 is and is the of the the role of Ser185 we of human PGI to phosphorylated Ser185 human PGI the of the and and changes the of human PGI phosphorylated at the of Ser185 is to the of the dimeric of it is that of the is phosphorylation. in the of the of and to the of the of changes the this we that human PGI/AMF is a that is phosphorylated at Ser185 by CK2 and that phosphorylation enzymatic activity. this PGI is as it with as kinase and in that phosphorylation enzymatic activity. PGI is in a of acid has whereas acid the enzyme to the the phosphorylation of PGI has also PGI Ser185 to and Ser185 in and that PGI phosphorylation is a isomerase glycolysis and and PGI as a protein that is the and it as which is in and a which the of and a which to and a PGI is in and in and is of associated with progression has that PGI phosphorylation is associated with revealed that Ser185 phosphorylation of human PGI the of the that phosphorylation with with of the sugar is by the that phosphorylation to the phosphorylation of the protein and that phosphorylation the of the protein that in the the PGI enzymatic by Ser185 phosphorylation the of Ser185 this is that Ser185 is the of the a phosphorylation changes by at the enzymatic of changes by the phosphorylation abolish the enzymatic of phosphorylated by the negative charge of the the of the also the enzymatic by the negative of the of Ser185 and the the of the in the is also that the negative charge by phosphorylation the of the at Ser185 a that by or a of at the the of PGI associated with and that the PGI that and that PGI the this that phosphorylation of PGI is known that of glycolysis with and in in and protein and the enzyme of the is in a PGI to and PGI the and metabolism in a PGI to the of that cellular and the that phosphorylation of PGI enzymatic and that this to to the decreased enzymatic of phosphorylated PGI changes in or and the mutant PGI proteins generated in analyses of changes in metabolism the of the this we that human PGI/AMF is a that is phosphorylated at Ser185 by CK2 and that phosphorylation enzymatic activity. this PGI is as it with as kinase and in that phosphorylation enzymatic activity. PGI is in a of acid has whereas acid the enzyme to the the phosphorylation of PGI has also PGI Ser185 to and Ser185 in and that PGI phosphorylation is a isomerase glycolysis and and PGI as a protein that is the and it as which is in and a which the of and a which to and a PGI is in and in and is of associated with progression has that PGI phosphorylation is associated with revealed that Ser185 phosphorylation of human PGI the of the that phosphorylation with with of the sugar is by the that phosphorylation to the phosphorylation of the protein and that phosphorylation the of the protein that in the the PGI enzymatic by Ser185 phosphorylation the of Ser185 this is that Ser185 is the of the a phosphorylation changes by at the enzymatic of changes by the phosphorylation abolish the enzymatic of phosphorylated by the negative charge of the the of the also the enzymatic by the negative of the of Ser185 and the the of the in the is also that the negative charge by phosphorylation the of the at Ser185 a that by or a of at the the of PGI associated with and that the PGI that and that PGI the this that phosphorylation of PGI is known that of glycolysis with and in in and protein and the enzyme of the is in a PGI to and PGI the and metabolism in a PGI to the of that cellular and the that phosphorylation of PGI enzymatic and that this to to the decreased enzymatic of phosphorylated PGI changes in or and the mutant PGI proteins generated in analyses of changes in metabolism the of the