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First published online August 31, 2007
Journal of Experimental Biology 210, 3295-3300 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006536
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p38 MAPK is a likely component of the signal transduction pathway triggering rapid cold hardening in the flesh fly Sarcophaga crassipalpis

Yoshihiro Fujiwara* and David L. Denlinger

Department of Entomology, Ohio State University, 400 Aronoff Laboratory, 318 West 12th Avenue, Columbus, OH 43210, USA


Figure 1
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  		Fig. 1. Comparison of predicted amino acid sequences of the phosphorylation domains of Sarcophaga p38 MAPK with Glossina, Drosophila and human kinases. The plus signs (+) indicate conserved amino acid residues that are phosphorylated. Asterisks (*) and periods (.) indicate identical and similar amino acids, respectively. The DDBJ, EMBL and GenBank accession numbers of these sequences are: AB277828 (Sarcophaga), ABC25081 (Glossina), AAC39030 (Drosophila p38a), AAC39032 (Drosophila p38b) and Q16539 (human p38a).

 

Figure 2
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  		Fig. 2. Temperature-dependent activation of p38 MAPK. Red-eye stage nondiapausing pharate adults were exposed for 2 h at the indicated temperatures. Fly proteins were analyzed using western blotting with anti-phospho-p38, anti-phospho-ERK, anti-phospho-JNK and anti-total p38 MAPK antibodies.

 

Figure 3
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  		Fig. 3. Temporal profiles of activation and decay of p38 MAPK phosphorylation in red-eye stage nondiapausing pharate adults. (A) Exposure to 0°C for various durations. (B) Exposure to 0°C for 10 min and then transferred to 25°C. (C) Exposure to 0°C for 2 h and then transferred to 25 and –10°C for 2 h, or held for an additional 2 h at 0°C. (D) Exposure to 0°C for 2 h and then transferred to 25°C for various durations. Flies held at 25°C were used as controls. Fly proteins were analyzed using western blotting with anti-phospho-p38 and anti-total p38 MAPK antibodies.

 

Figure 4
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  		Fig. 4. Development-specific phosphorylation of p38 MAPK in nondiapausing flies at 0°C. Flesh flies were reared at a nondiapausing condition of (A) 25°C or (B) 20°C. Larvae or pupae were held at 0°C for 2 h, and protein samples were prepared. Fly proteins were analyzed using western blotting with anti-phospho-p38 and anti-total p38 MAPK antibodies. FL, 3rd-instar feeding larvae; W0-2, day 0–2 wandering 3rd-instar larvae; P0–12, day 0–12 after pupariation; NT, no treatment; 0°C, held at 0°C for 2 h. Red-eye stage nondiapausing pharate adults reared at 25°C were used as controls.

 

Figure 5
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  		Fig. 5. Phosphorylation of p38 MAPK in diapause-programmed flies reared at 20°C and exposed to 0°C. (A) Feeding 3rd instar to day of pupariation, (B) diapausing pupae 0 to 50 days after pupariation and (C) day 20 diapause pupae to which 5 µl hexane was applied to elicit diapause termination. Larvae or pupae were held at 0°C for 2 h, and protein samples were prepared. Whole body proteins were analyzed using western blotting with anti-phospho-p38 and anti-total p38 MAPK antibodies. FL, 3rd-instar feeding larvae; W0–3, day 0–3 wandering 3rd-instar larvae; P0–50, day 0–50 after pupariation; NT, no treatment; 0°C, held at 0°C for 2 h. Red-eye stage nondiapausing pharate adults reared at 25°C were used as controls.

 

Figure 6
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  		Fig. 6. Tissue-specific and head-independent phosphorylation of p38 MAPK at 0°C. (A) Red-eye stage nondiapausing pharate adults were exposed at 0°C for 2 h, and specific tissues were dissected. Tissue proteins were analyzed using western blotting with anti-phospho-p38 and anti-total p38 MAPK antibodies. (B) Red-eye stage nondiapausing pharate adults were ligated between the head and thorax with a nylon thread and exposed at 0°C. Protein samples from the posterior part were analyzed using western blotting with anti-phospho-p38 and anti-total p38 MAPK antibodies. NT, no treatment; 0°C, held at 0°C for 2 h.

 

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© The Company of Biologists Ltd 2007