Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster
Abstract
:1. Introduction
2. Results
2.1. The foraging Gene Encoded Eight Putative Protein Isoforms that Differed at Their N-Termini
2.2. Many FOR Polypeptides Were Observed In Vivo and Their Expressions Differed between Developmental Stages
2.3. The Expression of FOR Differed between Tissues
2.4. The Majority of Whole-Body FOR Expression Was P1, P1α, and P3
2.5. LC-MS/MS of FOR Bands Corroborated Their Isoform Identity
2.6. LC-MS/MS Revealed Conserved Phosphorylation of the Active Loop Residues in FOR
2.7. FOR Sequence Differed at Several Amino Acids in fors and forR
2.8. P7 Was a Putative FOR Isoform Specific to fors
2.9. FOR Expression Differed between fors and forR Larvae
3. Discussion
4. Materials and Methods
4.1. Fly Strains and Rearing Conditions
4.2. Recombineering the forBAC::HA Allele
4.3. FOR Isoform-Specific Antibodies
4.4. Dissections
4.5. SDS-PAGE and Western Blotting
4.6. Immunoprecipitations and Mass Spectrometry
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Antibody | Antigen Sequence | Isoform Specificity |
---|---|---|
Anti-FOR(1) | SPPKIKENLSKSSSAYSTFSSAAEDSQDQVVICQQPQ (P2 residues 191 to 227) | P2, P4 |
Anti-FOR(2) | RRLSLEQAIEGLKLEGEKAVRQKSPQISPAASSNGSSKDLNGEGFCIPRPRLIVPVHTYARRRRTGNLKEQSSGGQE (P2 residues 286 to 362) | P2, P4 |
Anti-FOR(3) [34] | WFDGFYWWGLQNCTLEPPIKPAVKSVVDTTNFDDYPPDPEGPPP (catalytic domain) | P1, P1α, P2, P3, P4, P5, P6, P7 |
Anti-FOR(4) | HSSTTVDAPPRPADVDVATVPVATTAPPPQQPVSNLFYADYQKLQPAIIDRDWERDRDTDTDTRSEAKPPDIVEHIEPVEEQRQIHTQIQSPAEIQIQIPPTPPAPSIQIQIQQRYRRHSSAEDRNLNTRRNDSNITEALRKAASMQ (P1 residues 25 to 171) | P1 |
Anti-FOR(5) | QQELQLQQRYQQLQQLQAQTQGLYTSQGSPVLYHQPSPGSSQPVAIPGATCHSPTQLQPPNTL (P1 residues 278 to 340) | P1, P1α |
Anti-FOR(6) | ISGCTPSGTGGSATPSPVGLVDPNFIVSNYVAASPQEECFIQIIQAKELKIQEMQRALQFKDNEIAELKSHLDKFQSVFPFSRGSAAGCAGTGGASGSGAGGSGGSGPGTATGATRKSGQNFQRQRALGISAEPQSESSLLL (P1 residues 351 to 492) | P1, P1α, P3 |
CNS | Carcass | Fat Body | Intestine | |
---|---|---|---|---|
FOR isoform expression by for promoter Gal4 expression [17] | P1, P5, P7 * (pr1) P1, P5 (pr2) P1α, P3 (pr3) P2, P4, P6 (pr4) | P1α, P3 (pr3) P2, P4, P6 (pr4) | P1α, P3 (pr3) | P1, P5, P7 * (pr1) P1, P5 (pr2) P1α, P3 (pr3) P2, P4, P6 (pr4) |
FOR isoform expression by modENCODE RNA-seq by region (FlyBase) | P1/P1α/P3 (exon 4) P2/P4 (exon 7) | P1/P1α/P3 (exon 4) P2/P4 (exon 7) | P1/P1α/P3 (exon 4) P2/P4 (exon 7) | P1/P1α/P3 (exon 4) P2/P4 (exon 7) |
FOR isoform expression | P1, P1α, P3 | P1, P1α, P2, P3, P4 | P1α, P2, P3, P4 | No detectable expression |
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Vasquez, O.E.; Allen, A.M.; So, A.K.-C.; Nguyen, Q.H.; Krause, H.M.; Levine, J.D.; Sokolowski, M.B. Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster. Int. J. Mol. Sci. 2023, 24, 10219. https://doi.org/10.3390/ijms241210219
Vasquez OE, Allen AM, So AK-C, Nguyen QH, Krause HM, Levine JD, Sokolowski MB. Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster. International Journal of Molecular Sciences. 2023; 24(12):10219. https://doi.org/10.3390/ijms241210219
Chicago/Turabian StyleVasquez, Oscar E., Aaron M. Allen, Anthony K.-C. So, Quynh H. Nguyen, Henry M. Krause, Joel D. Levine, and Marla B. Sokolowski. 2023. "Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster" International Journal of Molecular Sciences 24, no. 12: 10219. https://doi.org/10.3390/ijms241210219