Signal Transduction Cascades and Phosphorylation Events

Protein Tyr phosphorylation increases during sperm capacitation, from about 2 h of incubation, and is often considered as a marker for this process [2, 17, 39, 44, 103, 104] . However, over the years, we described several transduction cascades forming a tightly regulated network of phosphorylation events, all regulated by ROS, that occur as earlier steps and are prerequisites for capacitation.

Soon after the onset of capacitation, there is a rise in cAMP levels [1, 2, 39, 43, 105, 106]; both exogenous O2'- and NO' can trigger this event [3, 17, 39, 43,

105, 107] maybe via activation of adenylyl cyclase [108] since phosphodiesterase activity does not seem to be modified (unpublished data) (Fig. 4.6). cAMP then stimulates PKA and a maximum activity is reached at 30 min [105]. This rise in PKA is translated to a rapid increase in the phosphorylation of two proteins (80 and 105 kDa) containing Ser and Thr residues within the Arg-X-X-(Ser/Thr) motif and collectively called phospho-PKA substrates [3, 17, 106]. In the boar, the equivalent two phospho-PKA substrates (59 and 96 kDa) obtained after differential extraction of the midpiece/large tail fraction appear to be members of the Odf2 (outer dense fiber) family of proteins [109]. This rise in phospho-PKA substrates also reaches a maximum at 30 min, occurs with various capacitation inducers (FCSu, progesterone, albumin, and FFu), including exogenous ROS (O,'-, H2O2, and NO'), and is prevented by SOD, catalase, NOS inhibitor (l-NAME), and PICA inhibitors (H89 and Rp-cAMP-S) [3, 17, 106] , Of interest, the rise in phospho-PKA substrates is also blocked by inhibitors of nonreceptor-type PTK (herbimycin A and PP2) but not when spermatozoa are stimulated with dbcAMP + isobutylmethylxanthine (IBMX, phosphodiesterase inhibitor), thus suggesting that a PTK acts upstream of PKA and maybe needed for adenylyl cyclase activation [3, 17, 106]. It is important to mention that this early-acting PTK is probably different of that responsible for the late Tyr phosphorylation of fibrous sheath proteins (p85 and p105). PKC, receptor-type PTK, or MEK does not appear to be involved in the regulation of the cAMP/PKA/ phospho-PKA substrates axis [3, 17, 106].

The ERK pathway then appears, in time, involved in human sperm capacitation (Fig. 4.6) [3, 5,17, 74, 79, 80, 110-112]. Most ofthe elements ofthe cascade, from Shc to Ras, as well as the ERK module common to all MAPK (mitogen-activated protein kinase) pathways [113, 114], and made of Raf (MAP kinase kinase kinase; phosphorylates Ser/Thr), MEK (MAP kinase kinase; dual specificity for Ser/Thr and Tyr), and ERK1 and ERK2 (MAP kinase; both for Ser/Thr), are present in human spermatozoa [74, 111, 112, 115, 116].

Sperm capacitation is associated not only with the phosphorylation of MEK (45 kDa; low amounts in human spermatozoa), but surprisingly also of MEK-like proteins (55, 94, and 115 kDa; higher levels) that reaches a maximum level at 60 min [112] (Fig. 4.6). The anti-phospho-MEK antibody recognizes these four proteins and PD98059 (inhibits MEK phosphorylation and activation) blocks their phospho-rylation, as well as capacitation, confirming these as MEK-like proteins [112].

Capacitation inducers, such as FCSu and BSA, as well as exogenous ROS (O2'_, H2O2, and NO'), promote the phosphorylation of MEK-like proteins and, conversely, SOD, catalase, and NOS inhibitor prevent this process emphasizing again the major role of ROS [112]. PKA inhibitors (H89 and Rp-cAMP-S) prevent the phosphorylation of MEK-like proteins [112] suggesting that PKA or phospho-PKA substrates are intermediate players involved in the cross talk between these phosphorylation pathway [3,17,112]. PKC, PTK (both nonreceptor and receptor type) and the ERK pathway also regulate the phosphorylation of MEK-like proteins [112].

Then, MEK (and MEK-like proteins) as dual-specificity kinase phosphorylates both Thr and Tyr residues within the Thr-Glu-Tyr motif present not only in ERK1 and ERK2 but also in several proteins, such as ERK5, ERK7, and MOK [117, 118].

Fig. 4.6 Proposed schema for phosphorylation events known to happen and be regulated by ROS during sperm capacitation. The main axes are presented: cAMP/PKA/P-PKA substrates (green), P-MEK and ERK cascade (black), PI3K/Akt (red), and late P-Tyr (blue). Blots (control and capacitating spermatozoa, left and right lines, respectively) and characteristic labeling pattern obtained with the various anti-phospho-antibodies are also presented. The time course of events is given on the right. ROS generated from the beginning of the capacitation period appear to act on adenylyl cyclase (AC) as one of the first targets and stimulate the production of cAMP. This cAMP activates PKA to phosphorylate its specific phospho-PKA (P-PKA) substrates. PKA and/or the P-PKA substrates might be involved in the phosphorylation of MEK-like (P-MEK-like) proteins and subsequently of Tyr residues (P-Tyr) of fibrous sheath proteins. PKA and the P-PKA substrates may also participate in other pathways associated with capacitation. A nonreceptor-type PTK, possibly activated by ROS, seems to modulate the increase in P-PKA substrates, maybe via activation of AC.

Fig. 4.6 Proposed schema for phosphorylation events known to happen and be regulated by ROS during sperm capacitation. The main axes are presented: cAMP/PKA/P-PKA substrates (green), P-MEK and ERK cascade (black), PI3K/Akt (red), and late P-Tyr (blue). Blots (control and capacitating spermatozoa, left and right lines, respectively) and characteristic labeling pattern obtained with the various anti-phospho-antibodies are also presented. The time course of events is given on the right. ROS generated from the beginning of the capacitation period appear to act on adenylyl cyclase (AC) as one of the first targets and stimulate the production of cAMP. This cAMP activates PKA to phosphorylate its specific phospho-PKA (P-PKA) substrates. PKA and/or the P-PKA substrates might be involved in the phosphorylation of MEK-like (P-MEK-like) proteins and subsequently of Tyr residues (P-Tyr) of fibrous sheath proteins. PKA and the P-PKA substrates may also participate in other pathways associated with capacitation. A nonreceptor-type PTK, possibly activated by ROS, seems to modulate the increase in P-PKA substrates, maybe via activation of AC.

Sperm capacitation is associated with a transient increase in the Thr-Glu-Tyr phosphorylation of ERK1 and ERK2 (p42 and p44) that peaks at 5 min [3, 74]. However, the Thr-Glu-Tyr phosphorylation of 27-33 kDa proteins (high for up to 2 h), as well as that of 80 and 105 kDa proteins (from 1 h and on) (Fig. 4.6), appears even more important because it is sustained and rises as capacitation proceeds [3, 74]. Inhibitors of MEK (PD98059 and U126) prevent capacitation and this phosphorylation confirming the ERK-like nature of these proteins [3, 74]. The double phosphorylation of the 27-33 kDa proteins depends on O2'- [74], but the role ofNO' is still unknown. The Thr-Glu-Tyr phosphorylation of 80 and 105 kDa proteins is also controlled by receptor-type PTK and the ERK pathway, but not by PKC or PKA [79, 80]. Of interest is that ROS regulation is this time (for 80 and 105 kDa proteins) limited to NO' . and not O.'- and H2O2 . which, in other words, means that NOS inhibitor prevents and exogenous NO' promotes this event [80]. This is, therefore, a very good example that ROS may act specifically on their targets.

Downstream, ERK1, ERK2, and ERK-like proteins phosphorylate substrates containing Ser or Thr within the motif proline (Pro)-X-X-Ser/Thr-Pro [114]. A monoclonal anti-phospho-Ser/Thr-Pro antibody (MPM2) [119] recognizes a doublet (78 and 80 kDa) of Triton-insoluble sperm proteins, the intensity of which increases with capacitation (Fig. 4.6) [74]. SOD and PD98059 block this phosphorylation, but the involvement of NO' is still unknown [3, 17, 74]. In the mouse, MPM2 antibody recognizes several sperm proteins (70-250 kDa), but the intensity of bands appears independent of MEK activity [110]. Therefore, the increased levels of various phosphorylations noted during capacitation are not only modulated by different ROS and kinases, but they may also vary from one species to the other.

The PI3K/Akt axis also participates in capacitation (Fig. 4.6). PI3K and Akt are involved in cell growth, proliferation, differentiation, etc. [120, 121] and also in sperm capacitation [3, 17, 102, 122' ' PI3K phosphorylates the phosphoinositide 3,4,5 (PIP3), an intermediate that activates, directly or via its downstream target Akt, different enzymes, such as PKC, PKA, and NOS [123]. The PI3K/Akt axis regulates

Fig. 4.6 (continued) The rise in P-MEK-like proteins that occurs 1 h after the beginning of capacitation is triggered by ROS. PKA, all the ERK pathway components, and PKC also modulate the rise of P-MEK-like proteins. We can hypothesize that H2O2 activates PKC, which in turn phospho-rylates Raf, the kinase normally responsible for the phosphorylation of MEK and MEK-like proteins. Inhibitors of PKA and of all elements of the ERK pathway prevent the increase in P-Tyr which allows suggesting that P-MEK-like proteins and P-PKA substrates are intermediates between the early events and the late increase in P-Tyr during capacitation. The phosphorylation of the Thr-Glu-Tyr motif in sperm proteins increases progressively from 1 h after the beginning of capacitation and is controlled by the entire ERK pathway and the PI3K/Akt axis. NO', but not O2'-or H'O2 ' triggers and modulates this phosphorylation. The PI3K/Akt axis is present in human spermatozoa and regulates the phosphorylation of the Thr-Glu-Tyr motif. PI3K, through its downstream effectors PDK1 and Akt, could activate NOS and the NO' formed could stimulate Ras and ERK pathway and later cause the increase in P-Tyr. Besides their effects on most of the kinases mentioned above, ROS may also inactivate several protein phosphatases (for Ser/Thr and for Tyr) and consequently prevent protein dephosphorylation at any step of capacitation (not shown on the schema to keep clarity)

capacitation and associated phosphorylation of Thr-Glu-Tyr and of Tyr on sperm proteins (FCSu or albumin as inducer) [102]. Furthermore, the phosphorylation of Arg-X-Arg-X-X-phospho-Ser/Thr, characteristic of Akt substrates, of several proteins (70, 80, 97, 110, and 140 kDa) increases during capacitation [102], but the modulation by ROS is presently not known. On the other hand, the Akt inhibitor prevents the rise of intracellular NO' formation in spermatozoa [56], suggesting that one of the effectors of Akt during capacitation could be NOS, specially considering that one of the phospho-Atk substrate is of 140 kDa [102], as NOS is [19, 43], Wortmannin (PI3K inhibitor) or the Akt inhibitor blocks Tyr and Thr-Glu-Tyr phosphorylation events [102] but not when capacitation is induced by exogenous H2O2 or NO- [5]; they do not affect the increases of either phospho-PKA substrates (unpublished data) or phospho-MEK-like proteins [5]. Therefore, the PI3K/Akt axis seems to modulate capacitation independently of the cAMP/PKA pathway but rather via an action on the ERK cascade. We could hypothesize that this is mediated by NO' since this ROS is known to activate Ras [124, 125],

Protein Tyr phosphorylation appears as a late event of capacitation occurring downstream of all other phosphorylation events mentioned above (Fig. 4.6) and is found in all species where it was studied, from rodents to domestic animals [65, 126-130], as well as in men [2, 17, 43, 130-132]. Tyr phosphorylation ofthe two Triton-insoluble proteins (81 and 105 kDa, antigenically related to the A-kinase-anchoring proteins, AKAPs) [44, 104, 130] is increased with all capacitation inducers tested (FCSu, FFu, BSA, progesterone, cell-permeant analogs of cAMP, etc.) as well as exogenous ROS (O2'-, H2O2, NO') and is prevented by SOD, catalase [2, 3, 17, 44, 130, 131], and inhibitors ofNOS [42, 130].

Several proteins are Tyr phosphorylated during capacitation beside the two well-known fibrous sheath proteins of 80 and 115 kDa (Fig. 4.6) [3, 17, 31, 111, 122, 130, 133, 134]. We can mention, between others, Triton-soluble proteins of 37, 42, and 47 kDa [14], AKAP 3, and valosin [135]. There are also several PTK, both of receptor [136] and nonreceptor type (Src, c-yes, Lyk, cAbl, etc.) [133,134,137-139], participating in these phosphorylation events. Although the association and/or role of both these PTK and their substrates in capacitation are indisputable, their regulation by ROS is not yet ascertained. We could expect that Src and Lyk are regulated by ROS as they are in other types of cells, but no data is actually available.

Therefore, ROS modulate all signal transduction cascades presently known to be related to capacitation, including cAMP/PKA, ERK pathway (MEK-like, ERK and ERK-like, and also ERK substrates), and PI3K/Akt axis [3, 17]. All these are needed and converge to the late downstream Tyr phosphorylation of two proteins of 80 and 105 kDa that was first thought to be modulated only by cAMP/PKA [131, 132].

The regulation of the pathways described above has to be looked at with a wide point of view since cross talks occur and the participation of other players is also needed. For example, PKC is one of those enzymes known to be directly activated by ROS, in part via the release of Zn2+ from this kinase [99, 140]. PKC is needed for capacitation and participates to the phosphorylation of the Thr-Glu-Tyr motif [79, 80], MEK-like proteins [112], and Tyr residues of fibrous sheath proteins (unpublished observations). Inhibition of PKC blocks the phosphorylation of MEK-like proteins triggered by FCSu or H2O2, suggesting that ROS generated during capacitation may activate PKC and one of its downstream effectors, Raf [3, 5, 17, 112, 141] . ROS appear to activate PKC and then Raf in bovine tracheal smooth muscle cells [141], supporting the possibility that this ROS-dependent stimulation of Raf through PKC may occur as well during human sperm capacitation.

PTKs, of both receptor and nonreceptor types, are susceptible of direct regulation through ROS [142] and are of paramount importance for the late Tyr phosphorylation of sperm proteins during capacitation [39, 42-44, 55]. It is conceivable that different PTKs must act considering the different time courses for the phosphoryla-tion of proteins, such as PKA substrates [106], MEK-like proteins [112], and those with the Thr-Glu-Tyr motif [79, 80], and of the late Tyr residues [39, 42-44, 55].

The specificity of ROS in their action should not be disputed even if in most cases O2'-, H2O2 , and NO. seem to have similar effects [2, 14, 40, 106, 112] since phosphorylation of the Thr-Glu-Tyr motif depends only on NO' [80] . Furthermore, ROS act, over time, at all steps of capacitation with activation of PKA [106] and Akt [102] atearly steps butthen also onphosphorylation ofMEK-likeproteins [112], of the Thr-Glu-Tyr motif [3, 5, 17, 74, 79, 80], and finally of Tyr residues. We have to remember also that NO' synthesis is essential for the whole course of capacitation [46], maybe because one of the downstream effectors for Akt is NOS.

In this section, we looked at how ROS modulate signal transduction cascades and pathways related to sperm capacitation and some of the cross talks that are involved. The next section reviews some of the mechanisms by which ROS can directly affect enzyme activity.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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