diminutive
Accurate identification of specific groups of proteins by their amino acid sequence is an important goal in genome research. This study combines information theory with fuzzy logic search procedures to identify sequence signatures or predictive motifs for members of the Myc-Max-Mad (see Drosophila Mnt for information about Mad family members) transcription factor network. Myc is a well known oncoprotein, and this family is involved in cell proliferation, apoptosis, and differentiation. A small set of amino acid sites from the N-terminal portion of the basic helix-loop-helix (bHLH) domain is described that provide very accurate sequence signatures for the Myc-Max-Mad transcription factor network and three of its member proteins. A predictive motif involving 28 contiguous bHLH sequence elements found 337 network proteins in the GenBank NR database with no mismatches or misidentifications. This motif also identifies at least one previously unknown fungal protein with strong affinity to the Myc-Max-Mad network. Another motif found 96% of known Myc protein sequences with only a single mismatch, including sequences from genomes previously not thought to contain Myc proteins. The predictive motif for Myc is very similar to the ancestral sequence for the Myc group estimated from phylogenetic analyses. Based on available crystal structure studies, this motif is discussed in terms of its functional consequences. The results provide insight into evolutionary diversification of DNA binding and dimerization in a well characterized family of regulatory proteins and provide a method of identifying signature motifs in protein families (Atchley, 2005).
c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs
(b-HLH-LZ). Binding to Max is essential for Myc transforming activity since Myc homodimers are inactive. Mutants of Myc and Max that bind efficiently
to each other but not to their wild-type partners were generated by either exchanging the HLH-leucine zipper domains
or reciprocally modifying LZ dimerization specificities. Complementary mutants are sufficient to cause transformation defects on their own, but they restore Myc
transforming activity when coexpressed in cells. The HLH-LZ exchange
mutants also produce the effect of dominant negative activity on wild-type Myc function. In addition, wild-type max
antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and
Myc-Max dimers for common target DNA sites. Therefore, Max can function as both suppressor and
activator of Myc. Amati (1993) discusses a general model for the role of Myc and Max in growth control.
Given that Myc family proteins appear to function through heterodimerization with the stable, constitutively expressed
bHLH-Zip protein, Max a lambda gt11 expression library was screened with radiolabeled Max proteinto determine whether Max mediates the function of regulatory proteins other than
Myc. One cDNA identified
encodes a new member of the bHLH-Zip protein family, Mad. Human Mad protein homodimerizes poorly but
binds Max in vitro, forming a sequence-specific DNA binding complex with properties very similar to those
of Myc-Max. Both Myc-Max and Mad-Max heterocomplexes are favored over Max homodimers, and, unlike
Max homodimers, the DNA binding activity of the heterodimers is unaffected by CKII phosphorylation. Mad
does not associate with Myc or with representative bHLH, bZip, or bHLH-Zip proteins. In vivo
transactivation assays suggest that Myc-Max and Mad-Max complexes carry out opposing functions in
transcription and that Max plays a central role in this network of transcription factors (Ayer, 1993a).
Mad is a basic-helix-loop-helix-zipper protein that heterodimerizes with Max in vitro. Mad-Max heterodimers
recognize the same E-box-related DNA-binding sites as Myc-Max heterodimers. However, in transient
transfection assays Myc and Mad influence transcription, albeit in opposite ways, through interaction with Max;
Myc activates while Mad represses transcription. Mad protein is induced rapidly
upon differentiation of cells of the myeloid lineage. The Mad protein is synthesized in human cells as a
35-kD nuclear phosphoprotein with an extremely short half-life (t1/2 = 15-30 min). It can be detected in vivo
in a complex with Max. In an undifferentiated monocyte cell line Max is found complexed with Myc
but not Mad. However, Mad-Max complexes begin to accumulate as early as 2 hr after induction of
macrophage differentiation with TPA. By 48 hr following TPA treatment only Mad-Max complexes are
detectable. Differentiation appears to be accompanied by a change in the composition of Max
heterocomplexes. This switch in heterocomplexes results in a change in the transcriptional
regulation of Myc-Max target genes required for cell proliferation (Ayer, 1993b).
Mxi1 belongs to the Mad (Mxi1) family of proteins, which function as potent antagonists of Myc oncoproteins. This
antagonism relates partly to the ability of these proteins to compete with Myc for the protein Max and for consensus DNA binding sites and to
recruit transcriptional co-repressors. Mad(Mxi1) proteins have been suggested to be essential in cellular growth control and/or
in the induction and maintenance of the differentiated state. Consistent with these roles, mxi1 may be the tumour-suppressor
gene that resides at region 24-26 of the long arm of chromosome 10. This region is a cancer hotspot, and mutations here may
be involved in several cancers, including prostate adenocarcinoma. Mice lacking Mxi1 exhibit progressive,
multisystem abnormalities. These mice also show increased susceptibility to tumorigenesis either following carcinogen
treatment or when also deficient in Ink4a. This cancer-prone phenotype may correlate with the enhanced ability of several
mxi1-deficient cell types, including prostatic epithelium, to proliferate. These results show that Mxi1 is involved in the
homeostasis of differentiated organ systems; it acts as a tumour suppressor in vivo, and engages the Myc network in a
functionally relevant manner (Schreiber-Agus, 1998).
Max on its own represses
transcription, whereas a significant stimulation is obtained when Max is coexpressed with c-Myc. Analysis
of specific mutants indicates that transcriptional activation requires both the c-Myc and the Max
dimerization and DNA-binding domains, as well as the c-Myc transactivation function; transcriptional
repression by Max requires both DNA binding and dimerization. Analogously, in stably transfected human
B-lymphoblastoid cell lines, overexpressed c-Myc and Max synergize to cause malignant transformation,
whereas overexpression of Max alone leads to growth inhibition. These results indicate that c-Myc and
Max are both transcriptional regulators, but their regulation of target-gene expression and cell proliferation takes place in opposite directions, most likely as the result of the opposite effects of heterodimeric c-Myc-Max (positive) versus
homodimeric Max (negative) complexes (Gu, 1993).
A method is described to design dominant-negative proteins (D-N) to the basic helix-loop-helix-leucine
zipper (B-HLHZip) family of sequence-specific DNA binding transcription factors. The D-Ns
specifically heterodimerize with the B-HLHZip dimerization domain of the transcription factors and
abolish DNA binding in an equimolar competition. Thermal denaturation studies indicate that a
heterodimer between a Myc B-HLHZip domain and a D-N consisting of a 12-amino acid sequence
appended onto the Max dimerization domain (A-Max) is more stable than the Myc:Max
heterodimer. One molar equivalent of A-Max can totally abolish the DNA binding activity of a
Myc:Max heterodimer. This acidic extension also has been appended onto the dimerization domain of
the B-HLHZip protein Mitf, a member of the transcription factor enhancer binding subfamily, to
produce A-Mitf. The heterodimer between A-Mitf and the B-HLHZip domain of Mitf is more stable than the Mitf homodimer. Cell culture studies show that A-Mitf can inhibit
Mitf-dependent transactivation both in acidic extension and in a dimerization-dependent manner.
A-Max can inhibit Myc-dependent foci formation twice as well as the Max dimerization domain
(HLHZip). This strategy of producing D-Ns may be applicable to other B-HLHZip or B-HLH proteins
because it provides a method to inhibit the DNA binding of these transcription factors in a
dimerization-specific manner (Krylov, 1997).
A recently identified mammalian histone
deacetylase (HD1) shows homology to the yeast Rpd3 (see Drosophila Rpd3) protein, which together with Sin3 affects the
transcription of several genes. Mammalian Sin3 proteins interact with the Mad components of the
Myc/Max/Mad network of cell growth regulators. Mad/Max complexes may recruit mammalian Rpd3-like
enzymes, thus directing histone deacetylase activity to promoters and negatively regulating cell
growth. A tetrameric complex composed of Max, Mad1, Sin3B
and HD1 is reported. This complex has histone deacetylase activity that can be blocked by the histone deacetylase
inhibitors trichostatin A and sodium butyrate. The inhibition of cell growth by Mad1 is enhanced by Sin3B
and HD1, as measured by colony formation assays. Furthermore, a Mad1-induced block of S-phase
progression can be overcome by trichostatin A, as shown in microinjection experiments.
It is concluded that the recruitment of a histone deacetylase by sequence-specific DNA-binding proteins
provides a mechanism by which the state of acetylation of histones in nucleosomes and hence the activity
of specific promoters can be influenced. The finding that Mad/Max complexes interact with Sin3 and
HD1 in vivo suggests a model for the role of Mad proteins in antagonizing the function of Myc proteins (Sommer, 1997).
X-ray structures of the basic/helix-loop-helix/leucine zipper (bHLHZ) domains of Myc-Max and Mad-Max heterodimers bound to their common DNA target (Enhancer or E box hexanucleotide, 5'-CACGTG-3') have been determined at 1.9 Å and 2.0 Å resolution, respectively. E box recognition by these two structurally similar transcription factor pairs determines whether a cell will divide and proliferate (Myc-Max) or differentiate and become quiescent (Mad-Max). Deregulation of Myc has been implicated in the development of many human cancers, including Burkitt's lymphoma, neuroblastomas, and small cell lung cancers. Both quasi-symmetric heterodimers resemble the symmetric Max homodimer, albeit with marked structural differences in the coiled-coil leucine zipper regions that explain preferential homo- and hetero-meric dimerization of these three evolutionarily related DNA-binding proteins. The Myc-Max heterodimer, but not its Mad-Max counterpart, dimerizes to form a bivalent heterotetramer, which explains how Myc can upregulate expression of genes with promoters bearing widely separated E boxes (Nair, 2003).
Genetic characterization of the promoters of putative myc regulated genes has provided further evidence for a physiological role for Myc-Max heterotetramerization. Oligonucleotide microarray analysis has identified several Myc target genes that contain multiple E boxes within promoters, typically separated by at least 100 nucleotides. Given the persistence length of DNA, this separation of Myc-Max binding sites is compatible with DNA looping stabilized by bivalent Myc-Max heterotetramers bound to two cognate sequences. Moreover, in vitro binding studies of Myc-Max heterodimers recognizing the lactate dehydrogenase gene, a natural high-affinity target containing two consensus E boxes, demonstrates that Myc-Max complexes can engage multiple cognate sites. Functional studies with adjacent E boxes found within the lactate dehydrogenase promoter have shown that mutations in either of the two E boxes severely affect Myc-dependent activation of transcription. These results suggest that Myc-Max complexes can form higher order structures that are consistent with the heterotetrameric assembly observed in the cocrystal structure (Nair, 2003).
Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. The subcellular locations of complexes formed by Myc/Max/Mad family proteins were visualized using bimolecular fluorescence complementation (BiFC) analysis. Max is recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 are enriched in nuclear foci, whereas complexes formed with Myc are more uniformly distributed in the nucleoplasm. Mad4 is localized to the cytoplasm when it is expressed separately, and Mad4 is recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 is determined by a CRM1-dependent nuclear export signal located near the amino terminus. The relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells were compared using multicolor BiFC analysis. Max forms heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it forms homodimers. Replacement of two amino acid residues in the leucine zipper of Max reverses the relative efficiencies of homo- and hetero-dimerization in cells. Surprisingly, Mad3 forms complexes with Max less efficiently than bMyc, whereas Mad4 forms complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms (Grinberg, 2004).
Transcription of the human proto-oncogene Myc is repressed in quiescent or non-dividing cells.
Upon mitogenic stimulation expression of Myc is rapidly and transiently induced, maintained
throughout G1, and then declines to a basal level throughout further cell cycle transitions. Regulation of
myc promoter activity critically depends on the presence of a binding site for transcription factor
E2F (Drosophila homolog: E2F). Transcription from the myc P2 promoter is induced
efficiently by E2F-1, but repressed by RB. Furthermore, overexpression of cyclin A strongly
activates the myc promoter and this effect is further enhanced by coexpression of E2F-1 and
cyclin A. Expression of G1-phase cyclin D1 leads to an E2F binding
site-dependent trans-activation of the myc promoter and this activation can be abrogated by
overexpression of RB. The interaction of D-type G1 cyclins with RB resembles that of the
adenovirus E1A protein with RB: both can disrupt inhibitory E2F-RB complexes. These results
support a model in which intervention of distinct cyclins and their respective associated kinases
promote transcriptional activation of myc throughout the cell cycle either by conversion of E2F
within multimeric complexes into an active transcription factor or by liberation of free functional
E2F (Oswald, 1994).
Genomic sequences were isolated flanking the 5' region of the E2F2 coding sequence. Various assays demonstrate promoter activity in this
sequence that reproduces the normal control of E2F2 expression during a growth stimulation. Sequence comparison reveals
the presence of a variety of known transcription factor binding sites, including E-box elements (that are consensus Myc binding
sites), as well as E2F binding sites. The E-box elements, which can function as Myc-responsive
sites, contribute in a positive fashion to promoter function. E2F-dependent negative regulation in quiescent
cells plays a significant role in the cell growth-dependent control of the promoter, similar to the regulation of the E2F1 gene
promoter (Sears, 1997).
Activation of the human cyclin E-cdk2 (See Drosophila homolog Cyclin E) heterodimer in quiescent cells involves a Myc-dependent step, but involves no significant change in the amount of cyclin-cdk complex. Such activation involves the release of a 120 kDa cyclin E-cdk2 complex from a 250 kDa complex, present in serum starved cells. The 250 kDa complex involves an association of cyclin-cdk with inhibitory molecule p27. Release of p27 involves either a change of affinity for p27 or p27 degradation. An additional step in activation of cyclin-dependent kinases by c-myc is dephosphorylation of cdk2 carried out by cdc25A (Drosophila homolog: string), a transcriptional target of Myc/Max heterodimers. Induction of cdc25A by Myc is direct, as it is
not inhibited by protein synthesis inhibitor cycloheximide. In the absence of adequate levels of growth factors, Cdc25A and myc share the ability to induce p53-dependent apoptosis. Myc-driven apoptosis is inhibited by cdc25A antisense oligonucleotides, suggesting that cdc25A expression might be essential for Myc-dependent apoptosis in some cells. The precise mechanism of cdc25A induction of apoptosis remains unclear (Steiner, 1995 and Galaktionov, 1996).
Ectopic expression of Mad1
inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1)
receptor. Mad1 also inhibits the ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F
(containing a Y-to-F mutation at position 809). Cell cycle analysis of proliferating
3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and
G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively
influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 that inhibit its activity as
a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1
to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor (Roussel, 1996).
Considerable evidence points to a role for G1 cyclin-dependent kinase (CDK) in allowing the
accumulation of E2F transcription factor activity and induction of the S phase of the cell cycle.
Numerous experiments have also demonstrated a critical role for both Myc and Ras activities in
allowing cell-cycle progression. Inhibition of Ras activity blocks the normal
growth-dependent activation of G1 CDK, prevents activation of the target genes of E2F, and results in
cell-cycle arrest in G1. Ras is essential for entry into the S phase in Rb+/+
fibroblasts but not in Rb-/- fibroblasts, establishing a link between Ras and the G1 CDK/Rb/E2F
pathway. However, although expression of Ras alone will not induce G1 CDK activity or S phase,
coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the
induction of S phase, coincident with the loss of the p27 cyclin-dependent kinase inhibitor (CKI). These
results suggest that Ras, along with the activation of additional pathways, is required for the generation
of G1 CDK activity, and that activation of cyclin E-dependent kinase in particular depends on the
cooperative action of Ras and Myc (Leone, 1997).
Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts
induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E
allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the
dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16.
Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation.
Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective
against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E
activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However,
cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of
E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with
effects indistinguishable from those of cyclin E. It is suggested that this effect of Myc is mediated by its
action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins,
rather than induction of Cdc25A. These data imply that oncogenic activation of c-Myc, and possibly also
of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis (Alevizopoulos, 1997).
Proto-oncogenes like c-myc are thought to control exit from the cell cycle rather than progression through the cell cycle itself. A different view of Myc function is presented in the current study. Exponentially growing Rat1-MycER fibroblasts were size-fractionated by centrifugal elutriation. In these cells, activation of cyclin E- and cyclin A-dependent kinases, degradation of p27, hyperphosphorylation of retinoblastoma protein and activation of E2F occur sequentially at specific cell sizes. However, upon activation of Myc, these transitions all occur simultaneously in small cells immediately after exit from mitosis. Interestingly, Myc has no discernible effect on the cell size at which DNA replication is initiated. Even in the presence of Myc activated cell cycle proteins, cells must grow to normal size before they initiate DNA replication and replication-dependent transcription of the histone H4 gene. These data show first that Myc controls the activity of G1 cyclin-dependent kinases independent of the transition between quiescence and proliferation or from any size effect on cell growth. These data also provide evidence of at least one dominant mechanism in addition to activation of E2F and of cyclin E/cdk2 kinase, which prevents DNA replication unless a critical cell size has been reached (Pusch, 1997).
Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, clonal MCF-7 breast cancer cell lines were derived in which c-Myc or cyclin D1 is expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 is sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression is not accompanied by increased cyclin D1 expression or Cdk4 activation, nor is cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 is sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21 following estrogen treatment. Interestingly, this is accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130 (a pRB-related pocket protein), identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis, which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21 (Prall, 1998).
Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. Biochemical and genetic evidence is provided to show that this sequestration is mediated via induction of cyclin D1 and/or cyclin D2 protein synthesis rates. Consistent with this conclusion, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant forming kinase-defective, Cki-binding cyclin-cdk complexes. At the same time Myc also induces cyclin E protein synthesis rate helping to promote a net gain of
newly formed Cki-free cyclin E-Cdk2 complexes. These become active concomitantly with phosphorylation of the kinase subunit by cyclin activating kinase (CAK). The sequestration function of D cyclins thus appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppresses cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescues the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation (Walker, 2005).
diminutive:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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