AKAP4 Protein Interactions and their Role in Anchoring a
Calcium Signaling Pathway in the Sperm Flagellum

Alexandre Daley and Regina Turner

University of Pennsylvania School of Veterinary Medicine

The overall objective of the work during the summers of 2002 and 2003 was to identify and characterize proteins which interact with pro-AKAP4 (formerly pro-AKAP82) to form a structurally and functionally normal sperm fibrous sheath.  The hypothesis is that the ‘pro’ domain of pro-AKAP4 anchors and organizes multiple components of a calcium signaling pathway in the sperm flagellum.

           AKAP4 and its precursor, pro-AKAP4, are major components of the horse, mouse and human sperm fibrous sheath (FS). Pro-AKAP4 is processed into mature AKAP4 by proteolytic cleavage of the amino-terminal ‘pro’ domain.   Historically, members of the AKAP family, including AKAP4, have been demonstrated to anchor protein kinase-A (PK-A) to a variety of subcellular locations.  In so doing, AKAPs direct the activity of the kinase and so increase its specificity of action. 

Prior work in the Turner lab had resulted in the isolation of over 100 DNA sequences that appear to interact with AKAP4, and/or its pro domain, in a Yeast 2 Hybrid assay. During the summer of 2002, we characterized these clones and identified sequences of particular interest. Initial work involved isolation, preparation and sequencing of plasmid clones containing potentially interacting inserts. Sequences were analyzed using a variety of online sequence databases and search engines (e.g. BLAST search, PROSITE, BLOCKS databases). Additionally, the insert size in each clone was determined using PCR and/or restriction enzyme digestion. Clones containing inserts over 300 bases in size and whose sequence suggested that they may be relevant to FS function or structure were transformed into E. coli for large-scale plasmid preparation.

The yeast 2-hybrid system yielded evidence that pro-AKAP4, in addition to anchoring PK-A, binds at least three putative members of a calcium signaling pathway to the sperm flagellum: calmodulin (CaM), a1H (a T-type calcium channel) and an uncharacterized EF-Hand domain containing protein (EFHP).   Calcium signaling has long been known to be involved in the regulation of several sperm functions including sperm motility and capacitation. 

A different calcium ion channel, CatSper1, has been identified in the sperm flagellum and has been shown to be critical for normal sperm motility.  Male mice with a targeted deletion of the CatSper1 gene are infertile and have poorly motile or immotile sperm.  Preliminary work with the yeast 2-hybrid system in the Turner lab suggests that CatSper1 also may be anchored to the flagellum via an interaction with pro-AKAP4. 

Thus, we now have preliminary evidence suggesting that pro-AKAP4 may act as a scaffold for at least two pathways central to the regulation of flagellar motility and sperm function:  PK-A and calcium signaling.  Our data suggests that each of these interactions occurs specifically via the ‘pro’ domain of the pro-AKAP4 protein.  However, because, false positive interactions can be detected by the yeast 2-hybrid system, there is a need to confirm these data using an independent method.  This year, relevant sequences were subcloned into expression vectors and expressed as fusion proteins containing either a His-Tag or an S-Tag.  We attempted to determine whether S-Tagged pro could be pulled down by His-Tagged CaM, a1H, EFHP, or CatSper1.  A positive pull-down would confirm the respective protein-protein interaction. 

            Sequences representing the relevant regions of CaM, a1H, the EF-hand domain containing protein and 2 overlapping regions of CatSper 1 were ligated into multicloning site 1 of the pACYDuet expression vector.  Sequences in this site are expressed as His-tag fusion proteins.  The sequence representing the ‘pro’ domain of pro-AKAP4 was ligated into multicloning site 2 of the pACYDuet expression vector for each of the above calcium pathway constructs.  Sequences in this site are expressed as S-tag fusion proteins. 

The pACYDuet vector allows for expression of 2 different proteins from a single vector within a single cell and so simplifies the study of protein-protein interactions.  Each construct was transformed into E. coli and fusion proteins were expressed routinely.  Cell extracts containing the His and S-tagged fusion proteins were then incubated in the presence of ProBond™ Nickel-Chelating Resin (Invitrogen) to allow binding of His-protein complexes to the resin.  After centrifugation and washing, bound proteins were eluted from the column using a low pH buffer as recommended by the manufacturer.  Eluted proteins were subjected to immunoblot analysis using both anti-His-tag and anti-S-tag antibodies.  This allowed us to determine whether or not each His-tagged protein was able to pull down ‘pro’-S-tag.  When available, the identity of bands representing each fusion protein was confirmed using protein-specific antibodies (antibodies against pro-AKAP4, ‘pro’, CaM and CatSper1 currently are available in the lab).

In the future, additional experiments will be performed to better characterize the potential functional significance of these interactions.

Alexandre Daley is a member of the Penn Vet Class of 2005.  She is working with Dr. Regina Turner for the summer as a research assistant.