Regulation of Schizosaccharomyces pombe Atf1 Protein Levels by Sty1-mediated Phosphorylation and Heterodimerization with Pcr1

Abstract

The Atf1 transcription factor plays a vital role in the ability of Schizosaccharomyces pombe cells to respond to various stress conditions. It regulates the expression of many genes in a stress-dependent manner, and its function is dependent upon the stress-activated MAPK, Sty1/Spc1. Moreover, Atf1 is directly phosphorylated by Sty1. Here we have investigated the role of such phosphorylation. Atf1 protein accumulates following stress, and this accumulation is lost in a strain defective in the Sty1 signaling pathway. In addition, accumulation of a mutant Atf1 protein that can no longer be phosphorylated is lost. Measurement of the half-life of Atf1 demonstrates that changes in Atf1 stability are responsible for this accumulation. Atf1 stability is also regulated by its heterodimeric partner, Pcr1. Similarly, Pcr1 levels are regulated by Atf1. Thus multiple pathways exist that ensure that Atf1 levels are appropriately regulated. Phosphorylation of Atf1 is important for cells to mount a robust response to H2O2 stress, because the Atf1 phospho-mutant displays sensitivity to this stress, and induction of gene expression is lower than that observed in wild-type cells. Surprisingly, however, loss of Atf1 phosphorylation does not lead to the complete loss of stress-activated expression of Atf1 target genes. Accordingly, the Atf1 phospho-mutant does not display the same overall stress sensitivities as the atf1 deletion mutant. Taken together, these data suggest that Sty1 phosphorylation of Atf1 is not required for activation of Atf1 per se but rather for modulating its stability. The Atf1 transcription factor plays a vital role in the ability of Schizosaccharomyces pombe cells to respond to various stress conditions. It regulates the expression of many genes in a stress-dependent manner, and its function is dependent upon the stress-activated MAPK, Sty1/Spc1. Moreover, Atf1 is directly phosphorylated by Sty1. Here we have investigated the role of such phosphorylation. Atf1 protein accumulates following stress, and this accumulation is lost in a strain defective in the Sty1 signaling pathway. In addition, accumulation of a mutant Atf1 protein that can no longer be phosphorylated is lost. Measurement of the half-life of Atf1 demonstrates that changes in Atf1 stability are responsible for this accumulation. Atf1 stability is also regulated by its heterodimeric partner, Pcr1. Similarly, Pcr1 levels are regulated by Atf1. Thus multiple pathways exist that ensure that Atf1 levels are appropriately regulated. Phosphorylation of Atf1 is important for cells to mount a robust response to H2O2 stress, because the Atf1 phospho-mutant displays sensitivity to this stress, and induction of gene expression is lower than that observed in wild-type cells. Surprisingly, however, loss of Atf1 phosphorylation does not lead to the complete loss of stress-activated expression of Atf1 target genes. Accordingly, the Atf1 phospho-mutant does not display the same overall stress sensitivities as the atf1 deletion mutant. Taken together, these data suggest that Sty1 phosphorylation of Atf1 is not required for activation of Atf1 per se but rather for modulating its stability. Eukaryotic cells have developed response mechanisms to combat the harmful effects of a variety of stress conditions. In the majority of cases, such responses involve changes in the gene expression pattern of the cell, leading to increased levels and activities of proteins that have stress-protective functions. Central to these responses are the sensing and signaling pathways that communicate with the nucleus and facilitate necessary changes in gene expression. Of particular importance are the pathways collectively known as MAPK 5The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ORF, open reading frame; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E3, ubiquitin-protein isopeptide ligase; MM, minimal medium. pathways (1Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (902) Google Scholar). The best characterized of these is a mammalian pathway that activates ERK1 and ERK2 in response to a variety of growth factors and mitogens and has been shown to be involved in the control of cell proliferation and differentiation (2Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar, 3Kyriakis J.M. Avruch J. BioEssays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar). Another subset of the MAPK pathways within a cell, the stress-activated protein kinases (SAPKs), responds to stress. In mammalian cells, two such pathways exist that lead to the activation of the JNK and p38 kinases (2Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar, 3Kyriakis J.M. Avruch J. BioEssays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar). A number of transcription factors are phosphorylated in response to SAPK activation, examples being c-Jun, which is regulated by JNK, and ATF2, which can be regulated by both JNK and p38 (4Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar, 5Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 6Livingstone C. Patel G. Jones N. 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The Sty1 MAPK stimulates gene expression via the Atf1 transcription factor, which is similar to the human ATF2 factor, and binds to closely related DNA sequences (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, 13Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.C. Toda T. J.B. Jones N. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). the transcription factor of the S. pombe pathway are closely related to a subset of phosphorylated and regulated by in mammalian cells. The Atf1 factor identified the S. pombe by a for that a in following and a for genes that the of cells (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, T. Toda T. K. A. M. Jones N. EMBO J. 1995; 14: PubMed Scopus Google Scholar, J. M. M. Genes 1996; PubMed Scopus Google Scholar). Atf1 binds to its with a protein Pcr1 M. Cell. Biol. 1996; PubMed Google the has a than N. S. A. PubMed Scopus Google Scholar). of the atf1 gene in a of in differentiation and of in and sensitivity to stress and stress T. Toda T. K. A. M. Jones N. EMBO J. 1995; 14: PubMed Scopus Google Scholar, J. M. M. Genes 1996; PubMed Scopus Google Scholar, G. Russell P. Cell. Biol. PubMed Google Scholar, A. K. Biol. Cell. PubMed Scopus Google Scholar, J. K. J.B. Jones N. Toone W.M. Biol. Cell. PubMed Scopus Google Scholar). Thus Atf1 is for cells to respond to a of stress conditions. have shown that activation of Sty1 in its to the nucleus binds and Atf1 G. K. Russell P. Genes Dev. PubMed Scopus Google Scholar). with the that the expression of Atf1 target genes Sty1 activation, that phosphorylation of Atf1 stimulates its the by which this is not In mammalian cells, of ATF2 by JNK phosphorylation has been investigated in to be a number of In the of ATF2 is to the DNA and the It has been that this is and activities ATF2 with such as c-Jun, is phosphorylated by Genes Dev. 1996; 10: PubMed Scopus Google Scholar). Phosphorylation has also been shown to the of the of ATF2, the involved C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (476) Google Scholar, 8van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (571) Google Scholar, S. D. B. Davis R.J. 1995; PubMed Scopus Google Scholar). of by the phosphorylation of ATF2 by to and N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. Biol. PubMed Scopus Google Scholar, I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this we to Atf1 be regulated phosphorylation. that phosphorylation regulates the stability of Atf1 as does with its Pcr1. however, to be responsible for the role of Sty1 in gene expression that of control exist that does not involve Atf1 phosphorylation. and pombe used are in and as S. A. P. PubMed Scopus Google Scholar). the cells a of a of and of the of various pombe used in this J. M. M. Genes 1996; PubMed Scopus Google and M. Cell. Biol. 1996; PubMed Google C. G. P. C. Nature. 1993; PubMed Scopus Google T. Toda T. K. A. M. Jones N. EMBO J. 1995; 14: PubMed Scopus Google K. M. Russell P. Biol. Cell. 9: PubMed Scopus Google in a of cells, not lead to activation of Sty1 not A has also shown that for not lead to a in the of gene expression with that cells by G. J. C.J. G. D. C. D. J. 4: PubMed Scopus Google with that does not the stress-activated MAPK pathway in to a of to protein of and atf1 the G. K. 1993; PubMed Scopus Google with two and the of the open reading by a and and by and and by a which the of the atf1 and the the A. PubMed Scopus Google Scholar). The with and the a wild-type strain and and number by and strain the of Atf1 Atf1. a of the atf1 to the ORF, and with the The a and the S. pombe J. M. M. Genes 1996; PubMed Scopus Google Scholar). as and S. pombe cell by in the following and protein cells in of cell by and to the of Pcr1 been by and by with and and used of in and in and used a of in M. M. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google used a of in and a used a of in a and used to the to the for of the and the and S. pombe the following A to of cells and by in of of the and for The cells for The by and the cells for a to The in of per of cells for protein and of cell protein used in the the in CHAPS, and to and the to the as for for for and for The by of the in the with a and to a for to the gene and with by of a DNA atf1 and levels to the the is the A. S. M. Biol. Google Scholar, T. D. K. Cell. Full Text Full Text PDF PubMed Scopus Google with of cells for with of and for cells with and in of by of in a of cells for a to of and of cell by of the cell as for with protein which with Atf1 with of of and of by of in of in a of for for in with and and for DNA the used not as as to for the of DNA to and the The of the gene used as used to the transcription to to and to for the to and to for the to and to for the to and to for the and to and to for the we of DNA a particular of of of and to the and the The of the to for a particular the following is the for the and and is the for the control Atf1 following Atf1 protein MAPK a by a the levels of Atf1 protein and two of stress, stress and stress to by to the mammalian ATF2 factor, phosphorylation Atf1 stability N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. Biol. PubMed Scopus Google Scholar, I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In wild-type cells the of Atf1 increased following stress In to increased the of the Atf1 protein this has been shown to be to phosphorylation (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, M. Cell. Biol. 1996; PubMed Google Scholar). are with the that stability of Atf1 is to its phosphorylation the and of phosphorylation to be dependent the particular of stress directly phosphorylation we a mutant atf1 gene single in MAPK gene the atf1 that atf1 expression the control of the the of stress, accumulation of the mutant protein observed The are with a of phosphorylation of the mutant protein and a phosphorylation for Atf1 accumulation. a in the of for the with wild-type but the levels not to to the that we observed in the of A in the of protein observed levels to and to a in the atf1 levels that we observed upon stress (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, G. V. J. Russell P. EMBO J. PubMed Scopus Google Scholar). we have the changes in gene expression in response to stress and in H2O2 stress, the levels of atf1 are and and of stress, in response to stress the levels of atf1 are and have to levels by D. Toone W.M. J. G. K. A. Jones N. J. Biol. Cell. 14: PubMed Scopus Google Scholar). are in with the we Atf1 protein levels in mutant a Sty1 pathway. is MAPK that and activates Sty1. In both and cells, the of Atf1 in the of stress lower than in wild-type cells no in levels and no of observed following H2O2 stress. In however, in cells, which have a of the MAPK K. M. Russell P. Biol. Cell. 9: PubMed Scopus Google Atf1 levels in the of stress and a a The levels not following stress. Thus the a the phosphorylation of Atf1 and its in the Atf1 and but MAPK the MAPK in Atf1 been important to the phosphorylation of because that Sty1 a that not to the MAPK A has shown that the mammalian of can of its a that does not the MAPK P. EMBO J. PubMed Scopus Google Scholar). we and cells and the Atf1 protein these and control The protein and of with to the and by the Atf1 protein in stress to be stress, as Atf1 In the protein not to be in the of stress. these the cells to The Atf1 protein cells of the same with multiple phosphorylated of Atf1. however, with to the because upon with observed by have also wild-type Atf1 protein a mutant. In this the Atf1 protein as a single by both in the of stress. the same Moreover, these the same as protein cells in the of of stress Taken together, these data are with not being a Atf1 upon in Atf1 levels following stress be to in atf1 gene in in of atf1 of Atf1 protein a of various protein stability plays a role in this we the half-life of Atf1 protein with stress. A a wild-type of the atf1 gene the control of the cells. in expression of Atf1 that is to of atf1 and of Atf1 protein by the of and and H2O2 to of the The levels of Atf1 protein by In the of stress, the half-life of Atf1 A and H2O2 however, a and in the half-life of Atf1 The levels of many ATF2, are regulated by Atf1 is also via a Atf1 levels in a mutant that a in the which a of the C. G. P. C. Nature. 1993; PubMed Scopus Google Scholar). the of and levels of Atf1 in this mutant the is that of wild-type cells M. C. K. J. Biol. 1996; PubMed Scopus Google Scholar). The of Atf1 the of Atf1 also in not The pattern of Atf1 phosphorylation upon stress not in the mutant data suggest that Atf1 is by the Taken together, the that the phosphorylation of Atf1 in increased accumulation of the Atf1 protein in to increased stability. these we the role of the MAPK phosphorylation upon Atf1 stability. A the the control of the cells. The levels of Atf1 protein following and with of cells a wild-type of the atf1 shown is than the wild-type Atf1 the of and levels a than wild-type Atf1 in the of stress that the phosphorylation of Atf1 by Sty1 is important for the stability of Atf1 as as stress conditions. with these Atf1 stability also in mutant cells Atf1 protein in with no in stability of Atf1 upon stress in cells Sty1 MAPK of the mutant protein and stability of Atf1 in a mutant demonstrates that the phosphorylation of Atf1 by Sty1 MAPK is responsible for the stability of Atf1 in both the and of 5The stability of Atf1 is regulated by the MAPK Sty1 and by its Pcr1. as in In in this the cells are for the atf1 gene but the mutant cells the used as the control cells with and and for Atf1 by In the H2O2 the of and the a mutant strain in the of H2O2 The stress the of and the in the Atf1 with the of this a pathway that the of Atf1 protein The of Atf1 protein in cells for with wild-type cells The Atf1 protein phosphorylated in the of Pcr1 as by a in its that is not a for phosphorylation of Atf1. Similarly, the levels of Pcr1 protein in cells for atf1 suggest that the two can a of ensure accumulation of in the cell and the accumulation of protein the which lead to the of this and cells with the a wild-type of the atf1 gene the control of the and in the of the of and Atf1 protein levels It is these data that Atf1 is in the of Pcr1. is the both in the of stress. upon H2O2 a in stability of Atf1 with the mutant in the of stress, which is to phosphorylation of Atf1. the Atf1 protein is in the mutant stress than in the wild-type in the of stress to have that Atf1 protein levels are by phosphorylation and by with Pcr1. is a of atf1 gene expression following the activation of Sty1 by a number of stress (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, G. V. J. Russell P. EMBO J. PubMed Scopus Google Scholar, D. Toone W.M. J. G. K. A. Jones N. J. Biol. Cell. 14: PubMed Scopus Google Scholar). of gene activation of transcription following with H2O2 G. V. J. Russell P. EMBO J. PubMed Scopus Google Scholar). the levels of to and upon for with H2O2 and D. Toone W.M. J. G. K. A. Jones N. J. Biol. Cell. 14: PubMed Scopus Google Scholar). The transcription of is shown as a its expression has been shown to be by Sty1 K. N. PubMed Scopus Google Scholar). The increased transcription of lost in for atf1 and and in the strain that the Thus expression dependent upon the Sty1 pathway and the Atf1 transcription factor the that the accumulation of Atf1 protein following stress is by in its Pcr1 a the two of the phosphorylation of Atf1 and the activation of and transcription has been shown (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, 13Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.C. Toda T. J.B. Jones N. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). the importance of Atf1 phosphorylation in stress responses has not been directly the stress sensitivity of cells atf1 to cells the in of the wild-type cells to mutant cells protein levels are protein be to these and stress conditions. the mutant than wild-type cells but its than that of cells Atf1. also suggest that the phosphorylation of Atf1 is for of growth stress conditions. we the response to stress this of of wild-type cells In of cells are In the of cells, the pattern of similar to that with wild-type cells. response to stress and stress to be defective in the mutant cells. A we the sensitivity of the mutant cells to levels of and Atf1 are required for the stress response with being for response to levels of H2O2 stress and Atf1 being required to respond to of these J. K. J.B. Jones N. Toone W.M. Biol. Cell. PubMed Scopus Google Scholar). mutant cells to levels of H2O2 as with cells, than wild-type cells Thus the to Atf1 does have a H2O2 in the also the expression of a number of genes in wild-type and mutant cells. of wild-type cells with H2O2 in activation of of the Atf1 target genes we In response to activation of target gene expression similar in wild-type and cells. activation in response to not the activation of both and lower in the mutant cells, expression in response to H2O2 the activation of target genes in the mutant activation that gene expression is by stress in the is that the in a of Atf1. shown in with the complete loss of activation of target genes the atf1 gene the gene is J.B. Buck V. Wilkinson M.G. Genes Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (311) Google Scholar, K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, 13Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.C. Toda T. J.B. Jones N. Genes Dev. 1996; 10: PubMed Scopus Google Scholar, G. K. Russell P. Cell. Biol. 1996; PubMed Scopus Google Scholar). Thus of Atf1 phosphorylation does have gene expression dependent upon the of stress and the of the target gene in no have we complete loss of which that Sty1 also by a that does not involve its phosphorylation. The activation of transcription in the mutant and stress be to transcription factors be the mutant Atf1 protein stress-dependent we to the protein of to the of genes. by Atf1 the of the Atf1 and proteins with the of two and both of which a Atf1 observed a in the Atf1 and of the to the and with the control which is not to be by Atf1. not observed in cells. a we the not of Atf1 to this data that phosphorylation of Atf1 is not required for this transcription factor to to its target is to to and the mutant displays sensitivity to The overall levels of induction of genes to be lower this stress The be a of loss of Atf1 phosphorylation as a of levels of Atf1 the the of protein in the cell be to the growth of the growth to the same because of wild-type with the sensitivity of the mutant to H2O2 to loss of protein rather than as a loss of phosphorylation In this we to the of phosphorylation upon Atf1. that in the MAPK phosphorylation as a the of Atf1 that is to phosphorylation lost. loss of Atf1 function not In to cells, the mutant not to a of stress and expression of a number of Atf1 target genes in a stress-dependent the protein to to its Pcr1. Wilkinson and N. a Atf1 is phosphorylated by and its ability to gene expression is phosphorylation per se not to be be required to this the of has been to be with stress The is a variety of transcription A. V. G. Cell. 7: Full Text Full Text PDF PubMed Scopus Google Scholar). can of the M. G. EMBO J. 10: Scopus Google Scholar, M. G. Nature. PubMed Scopus Google Scholar). The of does not phosphorylation but does the of a strain a mutant of with MAPK to expression of the gene upon stress, which is of with does not to be a however, as of in the transcription factor activation of a Cell. Biol. PubMed Scopus Google Scholar). the phosphorylation of ATF2, the mammalian of to be for its because a the two MAPK phosphorylation displays the same as ATF2 mutant. and N. It be to the role of Sty1 in its and the of the of target genes. is that Atf1 is in cells as a with factors that to its Phosphorylation of Atf1 these factors by Sty1 be to the in In this has been to the a with the of factors M. K. Cell. 9: Full Text Full Text PDF PubMed Scopus Google Scholar). that we in the stability of Atf1 following activation of the Sty1 pathway. stress Atf1 protein this is lost in a the Atf1 phosphorylation are Thus the in Atf1 levels is dependent upon Atf1 phosphorylation. The for the accumulation to be to in the half-life of the the be sensitivity to because Atf1 is in that are defective in Atf1 as a in (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, J. M. M. Genes 1996; PubMed Scopus Google Scholar). data that the phosphorylation to be of the MAPK because we not a the protein cells with of the levels of protein in cell with the stability that this phosphorylation to the Atf1 protein in the of stress. the protein is to as by the observed in in the of stress. The phosphorylation a of which is increased by upon stress. The levels of phosphorylation to the to which the is to with Atf1. In are the to the with and M. P. Curr. Opin. Genet. Dev. 10: PubMed Scopus Google Scholar). In the of phosphorylation has the and in Similarly, phosphorylation of the factor its with the N. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). to the responsible for Atf1 for and to phosphorylation this It be to the function of Sty1 phosphorylation identified to its also demonstrates that Atf1 levels are with the levels of its protein Pcr1. two proteins and to in the and of stress not In the of Atf1 levels to increased of Similarly, in the of Pcr1 levels Thus to be that in the two proteins that ensure that are both in the cell similar the of Atf1 Pcr1 such in the of the that such is the is of that for and which upon and the is than M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). gene expression is also the control of Atf1 and is following stress. suggest that this lead to levels of Pcr1 protein which lead to a of the in Atf1 levels to the of Atf1 levels Pcr1 and to the of in the of the activation of the Sty1 pathway is transcription leading to a in Pcr1 protein levels and as a Atf1 protein In addition, transcription of atf1 is and phosphorylation of Atf1 is which lead to increased Thus a of these mechanisms the levels of the can be and The of atf1 is also and regulated by the Sty1 (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, G. V. J. Russell P. EMBO J. PubMed Scopus Google Scholar). have shown that as as also levels stress, a of is by the which binds to atf1 and G. V. J. Russell P. EMBO J. PubMed Scopus Google Scholar). Sty1 is also required for the of the atf1 and upon to It is not however, these upon stress. this to be to stress, but does of by Sty1 the levels of Atf1 and Pcr1. does the mutant display sensitivity to H2O2 and not It is that this is to the that are than as many genes that Atf1 for increased transcription upon H2O2 for upon stress D. Toone W.M. J. G. K. A. Jones N. J. Biol. Cell. 14: PubMed Scopus Google Scholar). It is that in the of the of protein has a in the of genes is The that of the H2O2 is with this have shown that phosphorylation regulates the stability of but a role in does not to be for DNA activation of transcription per but does not that the pattern of phosphorylation of Atf1 with both and of stress J. K. J.B. Jones N. Toone W.M. Biol. Cell. PubMed Scopus Google Scholar). Thus be that the phosphorylated of Atf1 have in to role in to this C. K. C. K. and for Samuels for the of Atf1 and Pcr1 and for with