Identification of novel interacting proteins for hamartin and tuberin and a minimal region of hamartin necessary for its interaction with tuberin

Abstract

In order to find interacting partners for the 133 a.a. long evolutionarily conserved region of hamartin and the C‑CCD of tuberin, these regions were cloned in‑frame in the yeast two‑hybrid DNA‑BD vector pGBT9. The hamartin and tuberin clones were named as pGBT9‑Ham133a and pGBT9‑TubCCD2, respectively.

The yeast two‑hybrid screening of a mouse fetal brain library cloned in the DNA‑AD vector pACT2 with the pGBT9‑Ham133 clone as a bait identified 34 transformants. 2/34 transformants showed positive results for nutritional selection as well as α‑ and β‑galactosidase activity. The DNA sequence analysis of the inserts of the pACT2 clones from these two transformants showed homology to the mouse MRJ gene (GenBank accession no. AF035962).

The yeast two‑hybrid library screening of the above library with the pGBT9‑TubCCD2 clone as a bait yielded a total of 70 transformants. 20/70 transformants showed positive results for nutritional selection as well as α‑ and β‑galactosidase activity. Further sequence analysis of the inserts of the pACT2 clones from 9/20 transformants showed that MRJ and carboxypeptidase E (CPE, GenBank accession no. NM_013494) were the putative interacting proteins for the C‑CCD of tuberin.

In order to confirm the interactions of hamartin with MRJ and tuberin with MRJ and CPE by co‑immunoprecipitation (co‑IP), polyclonal antibodies were raised against the C‑terminal region of hamartin (a.a. 488–1016), N‑terminal region of tuberin (a.a. 235–462), C‑terminal region of MRJ (a.a. 60–242) and C‑terminal region of CPE (a.a. 136–476) by expressing these protein regions in bacteria and injecting the bacterially expressed and purified proteins in rabbits and rats. The specificity of the antibodies was checked by western blot analysis using adult mouse brain lysate.

The interaction of MRJ with hamartin, and of CPE and MRJ with tuberin, was confirmed under in vivo conditions by co‑IP experiments using adult mouse brain and human fetal brain lysates. Hamartin and tuberin showed interaction with both the isoforms of MRJ encoding for 27 and 37 kDa proteins in human.

As MRJ interacts with hamartin, MRJ and CPE interact with tuberin, and hamartin and tuberin are known to interact with each other, co‑IP assays were performed to see if MRJ and CPE interact with each other using adult mouse and human fetal brain lysates. The results showed that MRJ and CPE interact with each other also. CPE was found to interact with only the 27 kDa MRJ protein in human. CPE was also found to interact with hamartin in both species.

As a further confirmation of interaction among these proteins, co‑localization studies were carried out in HepG2 cells using immunofluorescence. MRJ was found to co‑localize with hamartin in the cytoplasm, whereas it was found to co‑localize with tuberin in the cytoplasm as well as in the nucleus. CPE was found to co‑localize with hamartin and tuberin in the cytoplasm only. MRJ and CPE were found to co‑localize in the cytoplasm.

As these four proteins interact with each other, it was reasoned that their expression should go hand in hand during different developmental stages of mouse. Using western blot analysis, the concomitant expression of all the four proteins was seen during all the mouse embryonic stages tested (day 10 to day 20) and postnatal day 2 mouse.

In case of CPE, in addition to the expected size 50 kDa band, an additional band of 60 kDa was also found to be expressed from embryonic day 10 to day 20 as well as in the brain of postnatal day 2 mouse. However, the 60 kDa band was absent in adult mouse brain lysate.

The western blot analysis using lysates from different tissues of adult mouse showed that the 60 kDa band of CPE was present in liver, spleen and lung lysates, while this band was absent in adult mouse brain and kidney lysates.

Further, in order to find out if these interactions are phosphorylation‑dependent, effect of λ‑protein phosphatase (λ‑PPase) was studied by incubating human fetal brain lysate with λ‑PPase followed by co‑IP using specific antibodies. The results showed that the interaction of MRJ with tuberin and CPE was not phosphorylation‑dependent, whereas the interaction between MRJ and hamartin was found to be phosphorylation‑dependent. The interaction of CPE with tuberin and hamartin was found to be phosphorylation‑independent.

It has been suggested that the first seven heptad structures of the CCD of hamartin is sufficient for its interaction with tuberin. Experiments were carried out to find out if less than seven heptad structures of CCD of hamartin are sufficient for its interaction with tuberin using the yeast two‑hybrid system. Surprisingly, the present results showed that the CCD of hamartin is not required at all for its interaction with tuberin. Instead, a 19 a.a. long region (a.a. 653–671) of hamartin, 5′ to its CCD, is necessary for its interaction with tuberin.

An isoform of hamartin was serendipitously identified while cloning the hamartin fragment from a.a. 653–727 using adult mouse brain cDNA as a template. This isoform showed in‑frame deletion of 15 nucleotides from positions 2254–2268 (5 a.a. from positions 678–682). As three isoforms of hamartin have been reported earlier, the isoform without deletion was designated as isoform‑4, whereas isoform with deletion of five a.a. was designated as isoform‑5.

RT‑PCR analysis using cDNA templates from different tissues of adult mouse showed that both the isoforms of hamartin (isoform‑4 and isoform‑5) were present in brain, liver, kidney, spleen, lung, heart, skin and muscle.

In order to see the effect of the deletion of five a.a. on the interaction of hamartin with N‑CCD of tuberin, isoform‑5 was cloned in the DNA‑BD vector pGBKT7. This clone (pHOCCDL) was co‑transformed with the clone pTNCCD, which contains N‑CCD of tuberin, in the yeast strain AH109. The results of yeast two‑hybrid assays showed that in‑frame deletion of five a.a. in hamartin had no effect on its interaction with N‑CCD of tuberin.