Abstract
Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.
Highlights
The serine-threonine protein phosphatase, protein phosphatase 2A (PP2A), is constitutively expressed in all eukaryotes
Western blot analysis of soluble extracts of sperm isolated from proximal caput, distal caput, and caudal regions of the epididymis when probed with anti-PP2A antibody each showed one immunoreactive band at ~36 kDa, presumably due to PP2A
A mouse monoclonal antibody anti-PP2A 4B7 raised against the amino acid sequence (RGEPHVTRRTPDYFL) corresponding to residues 295–309 in human PP2A-C is demethyl-sensitive
Summary
The serine-threonine protein phosphatase, protein phosphatase 2A (PP2A), is constitutively expressed in all eukaryotes. The 36 kDa catalytic subunit (PP2A-C) is tightly bound to the 65 kDa A scaffolding subunit (PR65) forming a heterodimer (AC) This dimer binds to a regulatory (B) subunit from one of four gene families designated as B/PR55, B'/ PR61/B56, B'', and B′′′ to form a hetero-trimer (ABC). The B'' family includes the isoforms PR48, PR59 and the splice variants PR72 and PR130, while the B′′′ family includes striatin and SG2NA [2]. Both A and B subunits have been shown to play a role in controlling the substrate specificity of PP2A. The B subunits appear to determine substrate specificity and sub-cellular localization of PP2A [3,4,5,6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
