Abstract
Developing male germ cells are extremely sensitive to heat stress; consequently, anatomic and physiologic adaptations have evolved to maintain proper thermoregulation during mammalian spermatogenesis. At the cellular level, increased expression and activity of HSP70 family members occur in response to heat stress in order to refold partially denatured proteins and restore function. In addition, several kinase-mediated signaling pathways are activated in the testis upon hyperthermia. The p38 MAP kinase (MAPK) pathway plays an important role in mitigating heat stress, and recent findings have implicated the downstream p38 substrate, MAPKAP kinase 2 (MK2), in this process. However, the precise function that this kinase plays in spermatogenesis is not completely understood. Using a proteomics-based screen, we identified and subsequently validated that the testis-enriched HSP70 family member, HspA1L, is a novel substrate of MK2. We demonstrate that MK2 phosphorylates HspA1L solely on Ser241, a residue within the N-terminal nucleotide-binding domain of the enzyme. This phosphorylation event enhances the chaperone activity of HspA1L in vitro and renders male germ cells more resistant to heat stress–induced apoptosis. Taken together, these findings illustrate a novel stress-induced signaling cascade that promotes the chaperone activity of HspA1L with implications for understanding male reproductive biology.
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References
Almog T, Naor Z (2008) Mitogen activated protein kinases (MAPKs) as regulators of spermatogenesis and spermatozoa functions. Mol Cell Endocrinol 282(1–2):39–44
Atarod S, Turner B, Pearce KF, Ahmed SS, Norden J, Bogunia-Kubik K, Wang XN, Collin M, Dickinson AM (2015) Elevated level of HSPA1L mRNA correlates with graft-versus-host disease. Transpl Immunol 32(3):188–194
Baum JS, George JPS, McCall K (2005) Programmed cell death in the germline. Semin Cell Dev Biol 16(2):245–259
Ben-Levy R, Leighton IA, Doza YN, Attwood P, Morrice N, Marshall CJ, Cohen P (1995) Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. EMBO J 14(23):5920–5930
Daugaard M, Rohde M, Jaattela M (2007) The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett 581(19):3702–3710
Dix DJ (1997) Hsp70 expression and function during gametogenesis. Cell Stress Chaperones 2(2):73–77
Dix DJ, Allen JW, Collins BW, Mori C, Nakamura N, Poorman-Allen P, Goulding EH, Eddy EM (1996) Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc Natl Acad Sci U S A 93(8):3264–3268
Dix DJ, Allen JW, Collins BW, Poorman-Allen P, Mori C, Blizard DR, Brown PR, Goulding EH, Strong BD, Eddy EM (1997) HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes. Development 124(22):4595–4603
Doshi BM, Hightower LE, Lee J (2009) The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock. Cell Stress Chaperones 14(5):445–457
Dun MD, Aitken RJ, Nixon B (2012) The role of molecular chaperones in spermatogenesis and the post-testicular maturation of mammalian spermatozoa. Hum Reprod Update 18(4):420–435
Eddy EM (1999) Role of heat shock protein HSP70-2 in spermatogenesis. Rev Reprod 4(1):23–30
Engel K, Ahlers A, Brach MA, Herrmann F, Gaestel M (1995) MAPKAP kinase 2 is activated by heat shock and TNF-alpha: in vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade. J Cell Biochem 57(2):321–330
Erbse A, Mayer MP, Bukau B (2004) Mechanism of substrate recognition by Hsp70 chaperones. Biochem Soc Trans 32(Pt 4):617–621
Ewen K, Jackson A, Wilhelm D, Koopman P (2010) A male-specific role for p38 mitogen-activated protein kinase in germ cell sex differentiation in mice. Biol Reprod 83(6):1005–1014
Fan W, Gao XK, Rao XS, Shi YP, Liu XC, Wang FY, Liu YF, Cong XX, He MY, Xu SB, Shen WL, Shen Y, Yan SG, Luo Y, Low BC, Ouyang H, Bao Z, Zheng LL, Zhou YT (2018) Hsp70 interacts with mitogen-activated protein kinase (MAPK)-activated protein kinase 2 to regulate p38MAPK stability and myoblast differentiation during skeletal muscle regeneration. Mol Cell Biol 38(24)
Feng HL, Sandlow JI, Sparks AE (2001) Decreased expression of the heat shock protein hsp70-2 is associated with the pathogenesis of male infertility. Fertil Steril 76(6):1136–1139
Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J, Saklatvala J (1994) Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78(6):1039–1049
Hasson SA, Kane LA, Yamano K, Huang CH, Sliter DA, Buehler E, Wang C, Heman-Ackah SM, Hessa T, Guha R, Martin SE, Youle RJ (2013) High-content genome-wide RNAi screens identify regulators of Parkin upstream of mitophagy. Nature 504(7479):291–295
Huusko JM, Karjalainen MK, Graham BE, Zhang G, Farrow EG, Miller NA, Jacobsson B, Eidem HR, Murray JC, Bedell B, Breheny P, Brown NW, Bodker FL, Litterman NK, Jiang PP, Russell L, Hinds DA, Hu Y, 23andMe Research, Rokas A, Teramo K, Christensen K, Williams SM, Ramet M, Kingsmore SF, Ryckman KK, Hallman M, Muglia LJ (2018) Whole exome sequencing reveals HSPA1L as a genetic risk factor for spontaneous preterm birth. PLoS Genet 14(7):e1007394
Ito Y, Ando A, Ando H, Ando J, Saijoh Y, Inoko H, Fujimoto H (1998) Genomic structure of the spermatid-specific hsp70 homolog gene located in the class III region of the major histocompatibility complex of mouse and man. J Biochem 124(2):347–353
Kato K, Ito H, Iwamoto I, Lida K, Inaguma Y (2001) Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27. Cell Stress Chaperones 6(1):16–20
Lambert H, Charette SJ, Bernier AF, Guimond A, Landry J (1999) HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus. J Biol Chem 274(14):9378–9385
Lanneau D, Brunet M, Frisan E, Solary E, Fontenay M, Garrido C (2008) Heat shock proteins: essential proteins for apoptosis regulation. J Cell Mol Med 12(3):743–761
Lee JH, Han YS, Yoon YM, Yun CW, Yun SP, Kim SM, Kwon HY, Jeong D, Baek MJ, Lee HJ, Lee SJ, Han HJ, Lee SH (2017) Role of HSPA1L as a cellular prion protein stabilizer in tumor progression via HIF-1alpha/GP78 axis. Oncogene 36(47):6555–6567
Lima SB, Cenedeze MA, Bertolla RP, Filho PA, Oehninger S, Cedenho AP (2006) Expression of the HSPA2 gene in ejaculated spermatozoa from adolescents with and without varicocele. Fertil Steril 86(6):1659–1663
Lizama C, Lagos CF, Lagos-Cabre R, Cantuarias L, Rivera F, Huenchunir P, Perez-Acle T, Carrion F, Moreno RD (2009) Calpain inhibitors prevent p38 MAPK activation and germ cell apoptosis after heat stress in pubertal rat testes. J Cell Physiol 221(2):296–305
Lu Z, Cyr DM (1998) Protein folding activity of Hsp70 is modified differentially by the hsp40 co-chaperones Sis1 and Ydj1. J Biol Chem 273(43):27824–27830
Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62(6):670–684
Naaby-Hansen S, Herr JC (2010) Heat shock proteins on the human sperm surface. J Reprod Immunol 84(1):32–40
Niederberger C, Agulnik AI, Cho Y, Lamb D, Bishop CE (1997) In situ hybridization shows that Dazla expression in mouse testis is restricted to premeiotic stages IV-VI of spermatogenesis. Mamm Genome 8(4):277–278
Nixon B, Bromfield EG, Dun MD, Redgrove KA, McLaughlin EA, Aitken RJ (2015) The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition. Asian J Androl 17(4):568–573
Paul C, Murray AA, Spears N, Saunders PT (2008) A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice. Reproduction 136(1):73–84
Radons J (2016) The human HSP70 family of chaperones: where do we stand? Cell Stress Chaperones 21(3):379–404
Rajapandi T, Wu C, Eisenberg E, Greene L (1998) Characterization of D10S and K71E mutants of human cytosolic hsp70. Biochemistry 37(20):7244–7250
Schlecht R, Erbse AH, Bukau B, Mayer MP (2011) Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat Struct Mol Biol 18(3):345–351
Scieglinska D, Krawczyk Z (2015) Expression, function, and regulation of the testis-enriched heat shock HSPA2 gene in rodents and humans. Cell Stress Chaperones 20(2):221–235
Son WY, Han CT, Hwang SH, Lee JH, Kim S, Kim YC (2000) Repression of hspA2 messenger RNA in human testes with abnormal spermatogenesis. Fertil Steril 73(6):1138–1144
Stokoe D, Caudwell B, Cohen PT, Cohen P (1993) The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2. Biochem J 296(Pt 3):843–849
Takahashi S, Andreoletti G, Chen R, Munehira Y, Batra A, Afzal NA, Beattie RM, Bernstein JA, Ennis S, Snyder M (2017) De novo and rare mutations in the HSPA1L heat shock gene associated with inflammatory bowel disease. Genome Med 9(1):8
Tsunekawa N, Matsumoto M, Tone S, Nishida T, Fujimoto H (1999) The Hsp70 homolog gene, Hsc70t, is expressed under translational control during mouse spermiogenesis. Mol Reprod Dev 52(4):383–391
Vydra N, Malusecka E, Jarzab M, Lisowska K, Glowala-Kosinska M, Benedyk K, Widlak P, Krawczyk Z, Widlak W (2006) Spermatocyte-specific expression of constitutively active heat shock factor 1 induces HSP70i-resistant apoptosis in male germ cells. Cell Death Differ 13(2):212–222
Williams PA, Krug MS, McMillan EA, Peake JD, Davis TL, Cocklin S, Strochlic TI (2016) Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis. Mol Biol Cell 27(15):2341–2350
Wisniewska M, Karlberg T, Lehtio L, Johansson I, Kotenyova T, Moche M, Schuler H (2010) Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B′, and HSPA5/BiP/GRP78. PLoS One 5(1):e8625
Yin Y, Hawkins KL, DeWolf WC, Morgentaler A (1997) Heat stress causes testicular germ cell apoptosis in adult mice. J Androl 18(2):159–165
Zhang M, Jiang M, Bi Y, Zhu H, Zhou Z, Sha J (2012) Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice. PLoS One 7(7):e41412
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We thank M. Krug for the generation of MK2-knockdown GC-2spd cells.
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Williams, P.A., Kobilnyk, H.E., McMillan, E.A. et al. MAPKAP kinase 2–mediated phosphorylation of HspA1L protects male germ cells from heat stress–induced apoptosis. Cell Stress and Chaperones 24, 1127–1136 (2019). https://doi.org/10.1007/s12192-019-01035-6
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DOI: https://doi.org/10.1007/s12192-019-01035-6