Post-Translational Modification of Human Heat Shock Factors and

Reviews pubs.acs.org/jpr

Post-Translational Modification of Human Heat Shock Factors and Their Functions: A Recent Update by Proteomic Approach Yan-Ming Xu,†,‡ Dong-Yang Huang,‡ Jen-Fu Chiu,§,¶ and Andy T. Y. Lau*,†,‡ †

Laboratory of Cancer Biology and Epigenetics, ‡Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041, China § The Open Laboratory for Tumor Molecular Biology, Department of Biochemistry, Shantou University Medical College, Shantou, Guangdong 515041, China ¶ Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China ABSTRACT: Heat shock factors (HSFs) are vital for modulating stress and heat shock-related gene expression in cells. The activity of HSFs is controlled largely by posttranslational modifications (PTMs). For example, basal phosphorylation of HSF1 on three serine sites suppresses the heat shock response, and hyperphosphorylation of HSF1 on several other serine and threonine sites by stress-activated kinases results in its activation, while acetylation on K80 inhibits its DNA-binding ability. Sumoylation of HSF2 on K82 regulates its DNA-binding ability, whereas sumoylation of HSF4B on K293 represses its transcriptional activity. With the advancement of proteomic technology, novel PTM sites on various HSFs have been identified with the use of tandem mass spectrometry (MS/MS), but the functions of many of these PTMs are still unclear. Yet, it should be noted that the discovery of these novel PTM sites provided the necessary evidence for the existence of these PTM marks in vivo. Followed by subsequent functional analysis, this would ultimately lead to a better understanding of these PTM marks. MS/MS-based proteomic approach is becoming a gold standard in PTM validation in the field of life science. Here, the recent literature of all known PTMs reported on human HSFs and the resulting functions will be discussed. KEYWORDS: heat shock factors, proteomics, post-translational modifications, mass spectrometry, tandem mass spectrometry, acetylation, phosphorylation, sumoylation, ubiquitylation

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INTRODUCTION The heat shock or stress responses in cells can only be efficiently modulated with the presence of heat shock factors (HSFs). HSFs are transcription factors that can bind specifically to heat shock promoter element (HSE), located upstream of heat shock protein (HSP) genes, which ultimately modulate heat shock-related gene expression in cells under stress conditions.1,2 As in the case of HSF1, at normal temperature, basal/ constitutive phosphorylation of HSF1 and the association with HSP90 complex repress HSF1’s transcriptional activity.3,4 Upon stress, basal level of HSPs in cells immediately mobilize and bind to the misfolded or partially denatured proteins, and as a result, HSF1 is relieved from inhibition and dissociated from HSP90 complex, allowing HSF1 to undergo trimerization and bind to HSE of HSP genes; various types of posttranslational modifications (PTMs) are further involved in regulating and attenuating the transactivation potential of HSF1, thereby controlling stress gene expression.1,2,5,6 © 2012 American Chemical Society

PTMs are among one of the critical determinants in modulating the activity of HSFs. In the past decades, despite the fact that in-depth investigations have been done on HSF1, the functions of other members in this family remain largely unexplored. With the inception of tandem mass spectrometry (MS/MS) and instrumentations, more and more unidentified or unexpected PTMs on HSF1 and other members in this family were gradually revealed.7−11 It is impossible to study PTMs comprehensively without these valuable tools. As a result of the competency of MS/MS technology, in recent years, a growing number of PTM sites of HSFs were identified by the use of this technology. Because of this, in this review, we would like to summarize the recent literature of all known PTMs on human HSFs and discuss the ultimate functions associated with these modifications. Received: November 21, 2011 Published: April 11, 2012 2625

dx.doi.org/10.1021/pr201151a | J. Proteome Res. 2012, 11, 2625−2634

2626

HSFY2

HSFY1

HSFX2

HSFX1

HSF5

Heat shock transcription factor, X-linked 1 (HSFX1) Heat shock transcription factor, X-linked 2 (HSFX2) Heat shock transcription factor, Y-linked 1 (HSFY1) Heat shock transcription factor, Y-linked 2 (HSFY2)

Heat shock transcription factor 2-like protein (HSF2L)

LW-1

Heat shock transcription factor 5 (HSTF5)




alternative protein name

Q96LI6

Q9UBD0

Q4G112

Q9ULV5

Q03933

Q00613

UniProtKB accession ID

203

214

isoform 3

401

475 423

24272

23370

45107

52541 46742

65278

49952

462 596

58293 53011

60348

52881

57260

mass (da)

518 492

isoform 2

isoform 1

isoform 2

isoform 2 isoform HSF4B* isoform HSF4A isoform 1

489

isoform short isoform 1 536

529

isoform long

isoform

length (amino acids)a

not yet confirmed

not yet confirmed

Y175ph • Y176ph

not yet confirmed K215su

not yet confirmed

not yet confirmed

not yet confirmed K293su • S298ph

K51ub • K82su • K139su • K151ub • K210ub • K420ub

M1ac • K80ac • S121ph • S127ph • T142ph • S195ph • S216ph • S230ph • S292ph • K298su • S303ph • S307ph • S314ph • S319ph • S320ph • T323ph • S326ph • S344ph • S363ph • T367ph • S368ph • T369ph • S419ph • S444ph not yet confirmed

amino acid modificationb

Methionine position as first codon. “isoform 1” or “isoform long” has been chosen as the “canonical” sequence, and all positional information refer to it, or otherwise the canonical form is indicated by an asterisk. Data were summarized from recent literature from NCBI and referenced from HGNC (HUGO Gene Nomenclature Committee) and UniProtKB (Protein knowledgebase of UniProt). b Nomenclature of all HSF PTMs also followed the new nomenclature for modified histone known as the Brno nomenclature.31 cThe protein existence of human HSF5 still needs to be confirmed.

a

Heat shock factor protein 4 (HSF4)

HSF4



HSF2

c


protein name

HSF1

gene name

Table 1. Properties and PTMs of Human Heat Shock Factors

Journal of Proteome Research Reviews


Journal of Proteome Research

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Figure 1. Schematic structure of human HSF family members. Functional domains are indicated as follows: the helix-turn-helix DNA-binding domain (DBD), the leuzine zipper oligomerization domain [heptad repeats (HR), HR-A, HR-B, and HR-C], the regulatory domain (RD), and the transactivation domain (TAD). The hydrophobic heptad repeats are important for controlling HSF from coiling to uncoiling during activation steps. The phosphorylation-dependent sumoylation motif (PDSM) is implicated in repressing the transactivating capacity of HSF1 or HSF4. Methionine position as first codon in all these sequences. Please note that human HSF5 has only been validated at transcript level and requires further confirmation. Data were summarized from recent literature from NCBI and referenced from HGNC (HUGO Gene Nomenclature Committee), UniProtKB (Protein knowledgebase of UniProt), and PhosphoSitePlus (http://www.phosphosite.org).

Mass Spectrometry-Based Proteomic Approach Accelerating the Confidence in PTM Determination

validated by MS analysis, further indicating the existence of these PTM marks in vivo and strengthening the credibility of the PTM data from previous non-MS-based studies.4,7,14,17 PTMs can also be identified by using only proteomic discovery-mode of mass spectrometric analysis. In this scenario, the subject being studied would be relatively complex and require a high-throughput, large-scale analysis, e.g., characterization of the cell nuclear phosphoproteins, quantitative atlas of mitotic phosphorylation, global SUMOylation (SUMO, small ubiquitin-like modifier) site determination, or quantitative assessment of the ubiquitin-modified proteome of the cells.8,9,18,19 The aim is to discover as many PTMs as possible that might present on these proteins in a global and aggressive way. Sometimes, unexpected PTMs were confirmed. For example, using this scenario, some previously uncharacterized in vivo PTM sites on HSFs were surprisingly identified after the data analysis, together with also the previously reported sites.8,9,11,19−21 However, it should be noted that drawbacks do occur, as it is often impossible to obtain some peptides bearing the PTMs (regardless of whether they are robust PTMs or less abundant PTMs) from a digest for MS/MS because these protein sequences contain multiple arginines or lysines close to and flanking the PTM site, which are readily cleaved by trypsin (usually the common protease used by many studies). As a result, some PTMs have never been covered in these MS studies so far and therefore remain unidentified, unless detailed top-down proteomics is employed. From the data of these studies, this also raises the possibility of underestimating the

In the past, the traditional way of studying protein PTM relied heavily on generating a site-specific modification-detecting antibody before the PTM under question can be validated at cellular level. In order to accelerate the speed and study protein PTMs unambiguously, mass spectrometry (MS)-based proteomic approach is the gold standard and the preferred approach. MS can circumvent the problems associated with the use of site-specific modification-detecting antibodies such as cross reactivity and epitope occlusion through interference by neighboring modifications.12,13 MS also has advantages over single modification-detecting antibody analysis, as it permits the simultaneous detection of multiple modifications on the same peptide and identification of unexpected modifications.7−11 Current Proteomic Approach in Deciphering Protein PTMs

In site-specific methods, usually the subject being studied is a low-throughput analysis and the PTMs under question are studied intensively. PTMs on the target protein were determined using amino acid sequencing, peptide mapping, site-directed mutagenesis, modification site-specific antibodies, and MS analysis.7,14−16 Nevertheless, MS analysis has its own advantage by providing convincing evidence for the existence of the PTMs where uncertainties might arise from conventional, indirect analysis (i.e., solely antibody-based analysis or sitedirected mutagenesis). For instance, many previously reported (non-MS-based studies) and novel PTM sites on HSF1 were 2627



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Figure 2. Representative cartoon of human HSF isoforms and their PTM sites. So far, only acetylation (ac), phosphorylation (ph), sumoylation (su), and ubiquitylation (ub) were identified on these transcription factors. Data were summarized from NCBI, HGNC, UniProtKB, and PhosphoSitePlus.

exact types and numbers of PTMs on these HSF proteins, and we can anticipate that there would be still an unidentified pool of PTMs waiting to be identified.

HSF4 consists of 492 amino acids for the canonical isoform HSF4B or 462 amino acids for isoform HSF4A. The region 245−319 of HSF4B (75 amino acids) is absent in HSF4A and replaced by a 45 amino acid region, making HSF4A shorter in length (30 amino acids) than HSF4B (Figure 2). Also, the PDSM is present in HSF4B (at position 292−299) but not in HSF4A. Human HSF4 has similar architecture as those of HSF1 and HSF2, but it lacks the HR-C, which could explain its constitutive trimerization and DNA-binding activity (Figure 1).23−25 Surprisingly, according to the data from UniProtKB, human HSF5 has only been validated at transcript level, while mouse HSF5 has been validated at protein level. If human HSF5 is really expressed at protein level, it should encode a protein of 596 amino acids for isoform 1 or 475 amino acids for isoform 2 (Figure 2). Also, a 121-amino acid region (53−173 of isoform 1) should be missing in isoform short (Figure 2). By homology analysis, the corresponding DBD would be located around region 10−200 (Figure 1). Although the protein existence of human HSF5 is obscure for the moment, Western blot analysis by using a commercial antibody against human HSF5 (abcam: ab98939), which is raised against a synthetic peptide, encompasses the C-terminal region from 546−595 of human HSF5, a band of around 60 kDa can be detected in ACHN human renal cell carcinoma cell lysate. This result shed light on the possible existence of human HSF5, at least in cancer cells at protein level. However, it is suggested that further studies such as by MS-based proteomic analysis be carried out to test this result. HSFX, also called LW-1, is X-linked and testis-specific. It has been found only on the X chromosome. Although two genes of HSFX, HSFX1 and HSFX2, exist on chromosome Xq28, they have the same cDNA sequence and encode the identical HSFX protein.1 Interestingly, HSFX is the only known HSF that has

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HUMAN HEAT SHOCK FACTORS To date, six types of human heat shock factors have been reported, namely, HSF1, HSF2, HSF4, HSF5, HSFX, and HSFY (Table 1 and Figure 1). HSF3 has only been recently identified in mice but not in humans.22 Various isoforms were expressed as a result from alternative splicing of the canonical transcripts (Figure 2). General Structure and Characteristics of Human HSFs

In general, one common feature of human HSF members is that they all consist of a helix-turn-helix DNA-binding domain (DBD) for sequence-specific DNA-binding to HSE (Figure 1). As in the most studied member HSF1, it consists of a DBD, several leucine zipper oligomerization domains [heptad repeats (HR), HR-A, HR-B, and HR-C], a regulatory domain (RD), and two transactivation domains (TAD) (Figure 1).2 The hydrophobic heptad repeats are important for controlling HSF from coiling to uncoiling during activation steps.1,2 The phosphorylation-dependent sumoylation motif (PDSM), I/L/ V-K-X-E-X-X-S-P, located at position 297−304 within the RD, is implicated in repressing the transactivating capacity of HSF1.15 The canonical long form of human HSF1 consists of 529 amino acids or 489 amino acids for isoform short. The sequence from position 462−489 is different between the two forms, and also the region 490−529 is missing in isoform short (Figure 2). HSF2 also exhibits similar structural architecture as that of HSF1 (Figure 1). It consists of 536 amino acids for the canonical isoform 1 or 518 amino acids for isoform 2. The region 393−410 is missing in isoform 2 (Figure 2). 2628



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Figure 3. Amino acid sequence alignment of human HSFX and HSFY isoforms. Alignments were done by Multalin interface (http://multalin. toulouse.inra.fr/multalin/). Red color indicates high consensus (90%), blue color for low consensus (50%), and black color for unaligned residues. Shown at the bottom is the consensus sequence of the subjects under comparison. Symbol “!” indicates homologous substituted amino acid residue I or V, “$” for L or M, and “#” for D, E, N, or Q. Dashes were inserted for optimal alignment.

and further phosphorylation at the RD by stress-activated protein kinases causes the full activation of HSF1 and activates heat shock gene expression.1,2 Moreover, once transcriptional activity is acquired upon stress, HSF1’s transcriptional activity is then abrogated during the attenuation phase. HSP70 and the cochaperone HSP40, induced from heat shock response, act as a negative feedback by interacting with DNA-bound HSF1, and this interaction inhibits the transactivation capacity of HSF1.1,2,5 Sumoylation of PDSM region within the RD represses the transactivating capacity of HSF1.15 HSF1 can also be regulated by the selective acetylation by the E1A-binding protein p300 (p300) (the transcriptional coactivator that plays pivotal roles in many physiological processes by acetylating diverse protein substrates), thereby releasing DNA-bound HSF1 and as a result inactivating the transactivation potential of HSF1.1,6 HSF1 was regarded as the principal stress-responsive regulator of heat shock response; however, HSF2 also binds to the promoters of HSP genes. It has been shown that HSF2 is able to modulate HSF1-mediated expression of HSP genes through heterocomplex formation with HSF1 and colocalize into nuclear stress bodies (NSBs).26,27 HSF1 is the most studied member among the HSF families. HSF1 is unable to bind to HSE sequence in the absence of stress. Although HSF2 showed common structural domains with HSF1, it is active at normal temperatures. HSF2 has been shown as a major constituent of constitutive binding to HSE in nonshocked mouse blastocysts, indicating that HSF2 might be involved in the control of heat shock gene expression during early mammalian embryogenesis.28 Interestingly, HSF4, the canonical isoform HSF4B, activates transcription. On the contrary, isoform HSF4A has been demonstrated to repress transcription, making HSF4A a natural corepressor for HSF4B.23−25 HSF4 has little role in the heat shock response, and it competes with HSF1 for common target genes in mouse lens epithelial cells and is involved in the development of different sensory organs in cooperation with HSF1.29,30

no isoforms and consists of 423 amino acids (Table 1, Figures 1 and 2). HSFY is Y-linked, testis-specific, and presents on the Y chromosome in Sertoli and spermatogenic cells. Two genes of HSFY, HSFY1 and HSFY2, exist on chromosome Yq11.221, and both have the same cDNA sequence and encode the identical HSFY protein.1 The canonical form of HSFY (isoform 1) consists of 401 amino acids, while isoforms 2 and 3 consist of 203 and 214 amino acids, respectively (Table 1, Figures 1 and 2). Besides being nearly half of the length of isoform 1, amino acid sequences are also replaced from region 172−203 for isoform 2, and region 173−214 for isoform 3 (Figure 2). The unusually small sizes of isoforms 2 and 3, which result from alternative splicing, somehow behave as two C-terminal truncated versions of isoform 1. For both human HSFX and HSFY, they consist of a DBD but do not have an obvious oligomerization domain (Figure 1); yet, they shared high amino acid sequence homology at the N-terminal (Figure 3). Besides human HSF1, all the other members in this family are still relatively understudied at present; it is expected that more functional studies will be carried out to delineate their precise domain organization and mechanism for transactivation. Functions of Human HSFs

In general, at normal temperature, HSF1 is a monomeric phosphoprotein that interacts with its major repressor HSP90 complex, which prevents HSF1 from undergoing homotrimerization.3,4 Basal/constitutive phosphorylation of HSF1 on three serine sites represses its transcriptional activity. Upon heat shock and in the attenuation phase, a number of protein kinases, acetylase, and SUMO-conjugating enzyme can influence the heat shock response in positive or negative ways by modifying HSF1 on distinct site(s).1,2 Upon heat shock, HSF1 is relieved from inhibition and dissociated from HSP90 complex; the severing of the intramolecular bond between HR-A and HR-B causes partial uncoiling of HSF1, which allows conformational change and homotrimerization of HSF1 and permits DNA-binding to HSE of HSP genes. The inactive trimers are then further uncoiled by breaking the bond between HR-B and HR-C, which is believed by the involvement of phosphorylation at the center of HR-B, 2629



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Table 2. Summary of PTM Marks Identified on Human Heat Shock Factors and the Resulting Functions (as of February 2012) PTM[ref]a HSF1 M1ac20 K80ac6 S121ph7,14

PTM[ref]

occurrence/function of the PTM

occurrence/function of the PTM

Protein trafficking of HSF1 Inhibits the DNA-binding ability of HSF1 Inhibition • Promotes molecular association with HSP90 • Upon heat shock treatment

S314ph7 S319ph7 S320ph35

S127ph47 T142ph34

Unknown Activation • Alters transcription • Regulates molecular association with HSF1

T323ph21,48,49 S326ph7

S195ph2 S216ph44,45

Activation Altered intracellular location • Connects HSF1 ubiquitylation • Protein degradation • Regulates cell cycle • Induces molecular association with Cdc20 Activation • Upon heat shock treatment Upon heat shock treatment Alters transcription • Represses its transcriptional activity and sumoylation on K298 is promoted by S303ph • Upon heat shock treatment Altered intracellular location • Alters transcription • Inhibition • Prerequisite for HSF1 sumoylation • Induces molecular association with 14-3-3 • Upon heat shock treatment Altered intracellular location • Alters transcription • Inhibition • Induces molecular association with 14-3-3 • Upon heat shock treatment

S344ph7 S363ph7,19,21,32

Upon heat shock treatment Upon heat shock treatment Activation • Altered intracellular location • Alters transcription • Upon heat shock treatment Unknown Activation • Alters transcription • Upon heat shock treatment Upon heat shock treatment Inhibition • Upon heat shock treatment

T367ph50 S368ph50 T369ph19,51

Unknown Unknown Unknown

S419ph7,16

Upon heat shock treatment • Control HSF1 nuclear translocation by heat stress

S444ph7

Upon heat shock treatment

Unknown Regulates DNA-binding of HSF2 Unknown

K151ub8 K210ub11 K420ub8

Unknown Unknown Unknown

Alters transcription • Represses its transcriptional activity and sumoylation on K293 is promoted by S298ph

S298ph15

Alters transcription • Inhibition • Prerequisite for HSF4 sumoylation

Y176ph10

Unknown

S230ph7,17 S292ph7 K298su15,36,37 S303ph7,15,19−21,32,33,36,40 S307ph7,19−21,33,40 HSF2 K51ub11 K82su52−54 K139su52−54 HSF4 K293su15 HSFX K215su9 HSFY Y175ph10

Unknown Unknown

a

Reference number(s) for the indicated PTM on HSFs are shown in superscript. Data were summarized from NCBI, HGNC, UniProtKB, and PhosphoSitePlus.

PTM would exert the same function in both forms has not been examined, and future proteomic approaches should interrogate separately the possible PTMs on both isoforms long and short. HSF1 has been reported to be acetylated, heavily phosphorylated on many serine and threonine residues, and sumoylated (Tables 1, 2 and Figure 2). However, no tyrosine phosphorylation or lysine ubiquitylation have been detected so far. It has been shown that phosphorylation on S121 [by MAPKactivated protein kinase 2 (MK2)],14 S303 [by glycogen synthase kinase 3 beta (GSK3β)],32,33 S307 [by extracellular signal-regulated kinase 1 (ERK1)],33 and S363 [by protein kinase C (PKC)]32 are inhibitory for transcription, whereas phosphorylation on T142 [by casein kinase 2-A2 (CK2-A2)],34 S195,2 S230 [by calcium/calmodulin-dependent protein kinase II (CaMKII)],17 S320 [by protein kinase A catalytic subunit alpha (PKAcα)],35 and S3267 are implicated in transcriptional activation. Phosphorylation on S121 by MK2 inhibits HSF1 transactivation and promotes HSP90 binding.14 S303 phosphorylation observed during heat shock can promote another modification by sumoylating K298, and phosphorylation on S303 is a prerequisite for HSF1 sumoylation on K298; these two PTMs are implicated in repressing the transactivating capacity of HSF1.15,36,37 Because of this S303ph-mediated K298 sumoylation, HSF1 is therefore regarded as the first example of PDSM-containing protein. The PDSM consensus sequence (i.e., I/L/V-K-X-E-X-X-S-P) lies at residues 297−304

Needless to say, the function of human HSF5 is still obscure for the moment because its existence at protein level is uncertain. The sex-linked genes, HSFX and HSFY, are also understudied transcription factors. Both of them shared high amino acid sequence homology at the N-terminal (Figure 3), suggesting a possible interplay among these players in the control of sex-linked-dependent gene regulation. PTM Marks Identified on Human HSFs and the Resulting Functions

For the sake of allowing patterns of HSF PTMs to be clearly and unambiguously specified, all the nomenclature of HSF PTMs also followed the new nomenclature for modified histone known as the Brno nomenclature.31 For example, phosphorylated HSF1 on serine 303 is abbreviated and presented as HSF1S303ph. So far, only acetylation (ac), phosphorylation (ph), sumoylation (su), and ubiquitylation (ub) on HSFs were reported (Tables 1, 2 and Figure 2); however, it is unclear if other types of PTMs are also utilized that govern the activity or stability of the HSFs. Future comprehensive MS-based proteomic approach for PTM determination would answer all these important questions. HSF1

By proteomic analysis, novel PTM sites on HSF1 were uncovered in recent years (Tables 1, 2 and Figure 2). Since these PTM sites were located on amino acid region 1−444, it is expected that these PTMs on isoform long should also occur in isoform short (Figure 2). However, whether the same types of 2630



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of HSF1 (i.e., V-K298-E-E-P-P-S303-P), is an important element within the RD of HSF1 for controlling its activity.15 Phosphorylation on S307 derepresses activation of heat stress, and in combination of S303, phosphorylation appears to be involved in recovery after heat-stress.4,38,39 Moreover, for phosphorylated S303 and S307, at least a portion of their inhibitory effect is due to recruitment of the phosphorylation-dependent binding protein 14-3-3 to HSF1S303ph and HSF1S307ph, resulting in the nuclear export of HSF1.40,41 Several modes of 14-3-3 binding motif typically exist: motifs for consensus binding included R-S-X-Sph/Tph-XP (mode 1) and R-X-X-X-Sph/Tph-X-P (mode 2), or simply just Sph/Tph for nonconsensus binding, where Sph/Tph indicates phosphorylated serine or threonine and X any amino acid except cysteine.42,43 However, regarding the consensus sequence recognition by 14-3-3, Winter et al. implicated that, as in the case of phosphorylated Histone H3 Ser10 and Ser28 (H3S10ph and H3S28ph) and 14-3-3 interaction, although the sequences do not completely fall in the consensus sequence recognized by 14-3-3, the binding of H3S10ph and S28ph to 14-3-3 is of high affinity. It has been shown that the proline 30 at position +2 of H3S28 (i.e., A-A-R-K-S28-A-P) is crucial for the high affinity binding of H3S28ph to 14-3-3.43 Although there is no proline at position +2 of H3S10 (i.e., T-A-R-K-S10T-G-G-K), the binding affinity of H3S10ph to 14-3-3 is somehow enhanced by other PTMs on vicinal amino acids surrounding H3S10, such as single acetylation on H3K9 or H3K14 (H3K9ac or H3K14ac). Therefore, 14-3-3 sometimes does not necessarily follow exactly its consensus sequence binding, and the affinity of 14-3-3 to phosphorylated substrate is varied among different substrates and local environments, since other factors may also involve in enhancing the association (such that high affinity binding can also occur for nonoptimal consensus sequence, which is compensated by other PTMs on vicinal amino acids). From the primary structure of HSF1, from residues 296 to 310 (i.e., R-V-K-E-E-P-P-S303-P-P-Q-S307-P-R-V), although the sequence surrounding S303 is not completely resembled to the mode 1 and mode 2 consensus-binding sequences recognized by 14-3-3 (Figure 4), similar to H3S28, there is a proline at position +2 of HSF1S303, which is believed as a crucial factor for efficient phosphoserine binding to 14-3-3.43 Although there is no proline at position +2 of HSF1S307, there is a possibility of compensation by other PTMs on vicinal amino acids surrounding HSF1S307 (such that high affinity binding can also occur for nonoptimal consensus sequence), which may enhance the ultimate phosphorylated HSF1−14-3-3 interaction. Besides, together with just nonconsensus phosphorylated serine-binding ability of 14-3-3 toward HSF1S303ph and S307ph, diverse modes of binding can actually be occurring (Figure 4). It has been shown that repression of HSP70B promoter activity by 14-3-3ε was effectively blocked by mutations at either or both of S303 and S307 sites of HSF1, suggesting that optimal HSF1 binding to 14-3-3ε requires HSF1 phosphorylation on S303 and S307, and serine-phosphorylation-dependent binding of HSF1 to 14-3-3ε results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm.40 In vivo, HSF1 is also repressed through phosphorylation on S363 by PKCalpha or -zeta but not MAPK.32 Therefore, regulation of HSF1 involves the action of three protein kinase cascades that repress HSF1 through phosphorylation on serine residues 303, 307, and 363 and may promote growth by suppressing the heat shock response. Acetylation on K80 by

Figure 4. Primary structure of human HSF1 and 14-3-3 protein− protein interaction. Although the sequence surrounding HSF1S303 is not completely resembled to the mode 1 and mode 2 consensusbinding sequences recognized by 14-3-3 (resembled region indicated by blue or red dashed open rectangle), the proline at position +2 of HSF1S303 is believed a crucial factor for efficient phosphoserine binding to 14-3-3.43 Together with just nonconsensus phosphorylated serine-binding ability of 14-3-3 toward HSF1S303ph and S307ph (black dashed open rectangles), diverse modes of binding are actually occurring. Both S303ph and S307ph of HSF1 are important for 14-3-3 interaction, and mutations at either or both of S303 and S307 sites of HSF1 abolished the interaction with 14-3-3.40.

p300 can also inhibit the DNA-binding ability of HSF1, while stress-induced deacetylation of this acetyl mark by the deacetylase sirtuin 1 (SIRT1) enhances the DNA-binding activity of HSF1.6 Phosphorylation on T142 by CK2 mediates transcriptional activity induced by heat.34 Lately, functional study by Calderwood et al. suggested that S195 phosphorylation at the center of HR-B is required for transactivation of HSF1 by breaking the bonds between HR-B and HR-C, in which inactive trimers are then uncoiled further.2 The fact that S195D mutation does not activate HSF1 at 37 °C suggests that this PTM is necessary but not sufficient for transactivation. S195A mutation inhibits the repressive effects of HSF1 on the c-f ms and c-fos promoters, while S195D mutation displays supportive effects.2 Phosphorylation on S230 by CaMKII promotes inducible transcriptional activity of HSF1.17 PKAcα is able to bind and phosphorylate HSF1 on S320, both in vitro and in vivo.35 Intracellular PKAcα levels and phosphorylation of HSF1 on S320 are both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of HSF1 target gene (hsp70.1), and activate hsp70.1 after stress. In 2005, by using MS and sequencing, Guettouche et al. demonstrated that heat treatment-activated HSF1 was phosphorylated on twelve serine residues: S121, S230, S292, S303, S307, S314, S319, S326, S344, S363, S419, and S444.7 More importantly, phosphorylation on S326 but none of the other serine residues was found to contribute significantly to the activation of the factor by heat and by oxidative stress agent cadmium chloride. Although heat stress-induced DNA-binding and nuclear translocation was not impaired in cells expressing the HSF1S326A substitution mutant, the stimulated HSP70 2631



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sequence (i.e., I/L/V-K-X-E-X-X-S-P) lies in residues 292−299 of HSF4B (i.e., L-K293-E-E-P-A-S298-P).15 Since HSF4A has different sequence at the region from 245−319 of HSF4B, these two PTMs are only written on HSF4B (Figure 2). This may likely explain the differential regulation or turnover of HSF4B and HSF4A in cells.

expression was several times less well than cells expressing the wild type HSF1. Mutagenesis and functional studies indicated that phosphorylation of HSF1 on S326 appears to be the final step in the remodeling of HSF1, which plays a key role in achieving the fully active trimer and transcriptional competence by stresses. However, no known functions of the other newly identified phosphorylated serines have been reported. It is believed that the observed heat-induced increase of phosphorylation on S303, S307, and S363 has a role in down-modulating HSF1’s activity during recovery from the stress. Besides, phosphorylation on S216 by Polo-like kinase 1 (Plk1) regulates cell cycle progression in which S216ph is implicated in binding with Cdc20 and the subsequent HSF1 ubiquitylation and degradation.44,45 Plk1 also phosphorylates HSF1 on S419 and mediates its nuclear translocation during heat stress.16 In addition, by the use of MS/MS, some novel PTMs were demonstrated to exist on HSF1 in vivo by large-scale analysis. Acetylation on the first codon methionine (M1) of HSF1 has been detected20 and may be related to protein trafficking since N-terminal acetylation has been shown to inhibit protein targeting to endoplasmic reticulum.46 Phosphorylation on S127,47 T323,21,48,49 T367ph,50 S368ph,50 and T369ph19,51 are also confirmed by quantitative phosphoproteomic studies, but the functions of these PTMs are currently unknown.

HSF5

Needless to say, the protein existence of human HSF5 would need to be validated by MS analysis first, prior to delineating the possible PTMs present on it. HSFX and HSFY

By MS/MS analysis from some studies, several novel PTMs on HSFX and HSFY were unintentionally discovered.9,10 Yet, the little piece of information from these studies also contributed to the expanding PTM list of HSFs. Although the functions of these PTMs are unknown, the results from these studies are valuable in that they provided the first evidence for these PTM marks in vivo and demonstrated the first few PTMs ever identified on HSFX and HSFY. It has been shown that HSFX is sumoylated on K2159 and HSFY isoform 1 is phosphorylated on Y175 and Y176 (Tables 1, 2 and Figure 2).10 HSFY is the only known HSF subjected to tyrosine phosphorylation so far, indicating potential upstream tyrosine kinase activity controlling HSFY’s function. Since HSFY isoforms 2 and 3 have different sequences from 172−203 and 173−214, respectively (and have no corresponding tyrosine residues as those in isoform 1), the two PTM marks are only written on isoform 1 (Figures 2 and 3). More importantly, although PTMs have not been reported in these replaced amino acid regions in isoforms 2 and 3, if they are, this may likely implicate the differential regulation or turnover among the three HSFY isoforms in cells.

HSF2

HSF2 isoform 1 has been reported to be sumoylated and ubiquitylated on distinct lysines (Tables 1, 2 and Figure 2). However, no phosphorylation or acetylation has been confirmed on HSF2 so far. Taking into consideration the high sequence homology between isoforms 1 and 2 of HSF2, all these sumoylation and ubiquitylation marks on isoform 1 would likely be also decorated on isoform 2 (Figure 2). It is yet to be determined whether these PTMs are present on both isoforms. K82 sumoylation of HSF2 results in conversion of this factor to the active DNA-binding form, which is the first demonstration that SUMO-1 modification can directly alter the DNA-binding ability of a transcription factor and reveals a new mechanism by which SUMO-1 modification can regulate protein function.52 Yet, the functional consequences of K82su are still unclear because sumoylation of HSF2 on K82 has been reported to both enhance and inhibit its DNA-binding ability.52−54 The HSF isoforms exhibit different sumoylation patterns that might provide complex physiological effects, whereas HSF1K298 is modified by either SUMO-1 or SUMO-2/ 3,15,37 HSF2K82 and K139 are modified by SUMO-1 and SUMO-2/3, respectively.52−54 Of these sites in HSF2, K82 is the primary sumoylation site, and K139 has been observed to be much less efficiently sumoylated than K82, and also the function of K139su is unknown.53 The functions of all the newly identified ubiquitylation sites, as proven to exist in vivo by MS/MS analysis (i.e., K51ub,11 K151ub,8 K210ub,11 and K420ub8), remain yet to be uncovered.

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PERSPECTIVES IN HSF PTMS AND FUNCTIONS BY PROTEOMIC APPROACH It should be noted that as more and more PTMs were identified on HSFs, the ultimate function of many of these PTMs still requires further intensive study. Therefore, we hope the reader would find the information here useful as a reference point. Suffice it to say, in the long run, MS/MS-based proteomic approach would gradually be able to interrogate all the possible PTMs on human HSFs and other transcription factors comprehensively. This would also be a gold standard in proving the existence of a particular PTM, whether it really occurs in vivo. The solid validation of the presence of PTM on proteins at the cellular level should be the first step before functional studies should be performed. This is critical and vital since without this in vivo validated information, any selected potential PTM sites being studied might actually not occur in cells, and the data obtained would lead to artifacts and wrong conclusions. PTM site determination by MS/MS analysis, followed by appropriate biochemical approaches, would certainly accelerate the progress in dissecting the multifaceted functions of transcription factors and allow us to understand what PTMs on transcription factors would govern their functions and what is the global meaning of these marks. Finally, we want to emphasize that with the advancement of MS/MS-based proteomic approaches for protein PTM determination, at least, we should begin to compare the whole PTM profile changes on a protein, i.e., inactivated versus activated state, normal versus diseased state, or the time course of PTM dynamics, as this would be meaningful toward

HSF4

So far, only sumoylation on K293 and phosphorylation on S298 have been confirmed on HSF4B (Tables 1, 2 and Figure 2). As mentioned, both HSF1 and HSF4B contain the PDSM, and sumoylation of HSF4B on K293 represses its transcriptional activity. Similar to the PDSM of HSF1, sumoylation of HSF4B on K293 is promoted by S298ph.15 The PDSM consensus 2632



Reviews

vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell. Proteomics 2011, 10 (10), M111.013284. (12) Garcia, B. A.; Barber, C. M.; Hake, S. B.; Ptak, C.; Turner, F. B.; Busby, S. A.; Shabanowitz, J.; Moran, R. G.; Allis, C. D.; Hunt, D. F. Modifications of human histone H3 variants during mitosis. Biochemistry 2005, 44 (39), 13202−13. (13) Lau, A. T. Y.; Xu, Y. M.; Chiu, J. F. Proteomic analysis of human pluripotent cells, In Human Stem Cell Technology and Biology: A Research Guide and Laboratory Manual; Stein, G. S., Borowski, M., Luong, M. X., Shi, M.-J., Smith, K. P., Vazquez, P., Eds.; John Wiley & Sons, Inc.: Hoboken, NJ, 2010; Chapter 21. (14) Wang, X.; Khaleque, M. A.; Zhao, M. J.; Zhong, R.; Gaestel, M.; Calderwood, S. K. Phosphorylation of HSF1 by MAPK-activated protein kinase 2 on serine 121, inhibits transcriptional activity and promotes HSP90 binding. J. Biol. Chem. 2006, 281 (2), 782−91. (15) Hietakangas, V.; Anckar, J.; Blomster, H. A.; Fujimoto, M.; Palvimo, J. J.; Nakai, A.; Sistonen, L. PDSM, a motif for phosphorylation-dependent SUMO modification. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (1), 45−50. (16) Kim, S. A.; Yoon, J. H.; Lee, S. H.; Ahn, S. G. Polo-like kinase 1 phosphorylates heat shock transcription factor 1 and mediates its nuclear translocation during heat stress. J. Biol. Chem. 2005, 280 (13), 12653−7. (17) Holmberg, C. I.; Hietakangas, V.; Mikhailov, A.; Rantanen, J. O.; Kallio, M.; Meinander, A.; Hellman, J.; Morrice, N.; MacKintosh, C.; Morimoto, R. I.; Eriksson, J. E.; Sistonen, L. Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1. EMBO J. 2001, 20 (14), 3800−10. (18) Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villén, J.; Li, J.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (33), 12130−5. (19) Dephoure, N.; Zhou, C.; Villén, J.; Beausoleil, S. A.; Bakalarski, C. E.; Elledge, S. J.; Gygi, S. P. A quantitative atlas of mitotic phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (31), 10762− 7. (20) Gauci, S.; Helbig, A. O.; Slijper, M.; Krijgsveld, J.; Heck, A. J.; Mohammed, S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal. Chem. 2009, 81 (11), 4493−501. (21) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127 (3), 635−48. (22) Fujimoto, M.; Hayashida, N.; Katoh, T.; Oshima, K.; Shinkawa, T.; Prakasam, R.; Tan, K.; Inouye, S.; Takii, R.; Nakai, A. A novel mouse HSF3 has the potential to activate nonclassical heat-shock genes during heat shock. Mol. Biol. Cell 2010, 21 (1), 106−16. (23) Nakai, A.; Tanabe, M.; Kawazoe, Y.; Inazawa, J.; Morimoto, R. I.; Nagata, K. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol. Cell. Biol. 1997, 17 (1), 469−81. (24) Zhang, Y.; Frejtag, W.; Dai, R.; Mivechi, N. F. Heat shock factor-4 (HSF-4a) is a repressor of HSF-1 mediated transcription. J. Cell. Biochem. 2001, 82 (4), 692−703. (25) Tanabe, M.; Sasai, N.; Nagata, K.; Liu, X. D.; Liu, P. C.; Thiele, D. J.; Nakai, A. The mammalian HSF4 gene generates both an activator and a repressor of heat shock genes by alternative splicing. J. Biol. Chem. 1999, 274 (39), 27845−56. (26) Sandqvist, A.; Björk, J. K.; Akerfelt, M.; Chitikova, Z.; Grichine, A.; Vourc’h, C.; Jolly, C.; Salminen, T. A.; Nymalm, Y.; Sistonen, L. Heterotrimerization of heat-shock factors 1 and 2 provides a transcriptional switch in response to distinct stimuli. Mol. Biol. Cell 2009, 20 (5), 1340−7. (27) Alastalo, T. P.; Hellesuo, M.; Sandqvist, A.; Hietakangas, V.; Kallio, M.; Sistonen, L. Formation of nuclear stress granules involves HSF2 and coincides with the nucleolar localization of Hsp70. J. Cell Sci. 2003, 116 (Pt 17), 3557−70.

understanding the basis of PTM decoration on the protein as a whole in a more global view.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 86-754-8853-0052. Fax: 86-754-8890-0437. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China Grants 31170785 and 81101785 (Andy T. Y. Lau) and Fund for University Talents of Guangdong Province (Andy T. Y. Lau). We would like to thank members of the Lau And Xu lab for critical reading of this review.

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ABBREVIATIONS HSF, heat shock factor; HSE, heat shock promoter element; HSP, heat shock protein; PTM, post-translational modification; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ac, acetylation; ph, phosphorylation; SUMO, small ubiquitinlike modifier; su, sumoylation; ub, ubiquitylation; PDSM, phosphorylation-dependent sumoylation motif

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REFERENCES

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Post-Translational Modification of Human Heat Shock Factors and

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