In vascular smooth muscle (VSM), contraction is initiated by phosphorylation of the regulatory light chain (RLC₂₀) of SM myosin II on Ser19 by the SM myosin light chain kinase (smMLCK, referred to hereafter as MLCK). MLCK is activated by Ca²⁺-bound calmodulin (CaM). (Gallagher et al., 1992; Hartshorne et al., 1998). In this way, Ca²⁺ influx promotes phosphorylation of RLC₂₀, activating SM myosin II and its binding to actin filaments, thereby initiating cyclic ATP-dependent cross-bridge cycling. The activity of MLCK is counterbalanced by that of the RLC₂₀ phosphatase (MLCP) which induces relaxation. MLCP itself is regulated by phosphorylation, specifically by the RhoA-dependent Ser/Thr kinase ROCK. The latter is activated by the small cytosolic GTPase RhoA, downstream of G-protein coupled receptors. This pathway sustains contraction as intracellular [Ca²⁺] decreases, a phenomenon known as Ca²⁺-sensitization (Somlyo and Somlyo, 2003).

The widely expressed mylk1 gene contains alternative promotors giving rise to three proteins: the long (∼220 kDa) non-muscle myosin II light chain kinase, expressed in various cell types; the short (∼130 kDa) isoform unique to SM acting on the myosin II isoform; and the non-catalytic kinase related protein also known as telokin (17 kDa) (Kamm and Stull, 2001; Herring et al., 2006). Telokin is a small protein whose sequence is identical to the COOH terminus of SM ∼220 and ∼130 kDa MLCKs and is independently expressed in SMs through a promotor located within an intron of the MYLK1 gene. Long form/short form/telokin will be used interchangeably with 220/130/17 kDa isoforms throughout the manuscript. In the classical view of SM contraction, MLCK was the sole protein kinase regulating RLC_20. Over the past few decades, strong evidence emerged that this scheme is oversimplified. For example, direct evaluation of MLCK activity in bladder SM from a CaM-sensor-MLCK transgenic mouse model demonstrated that only about 50% of carbachol-induced force and RLC₂₀ phosphorylation was accounted for by MLCK activity (Isotani et al., 2004). Thus, additional mechanisms exist for increasing both force and RLC₂₀ phosphorylation. The possibility of other auxiliary kinases regulating SM contractility is of enormous significance, because reactivity and tone of vascular SM affects vascular resistance, and consequently blood pressure (BP) (Bohr and Sitrin, 1970; Goulopoulou and Webb, 2014; Touyz et al., 2018). Studies in mice with tamoxifen-induced, SM-specific partial MLCK knockout have shown that BP is significantly reduced and salt-induced hypertension is abolished (He et al., 2008; He et al., 2011). However, therapeutic inhibition of MLCK is not a practical option, in part because the ubiquitously expressed non-muscle myosin shares the same catalytic domain and because of its major role in contractile activity of all SM-containing hollow organs. As a result, there is ongoing research to identify auxiliary, regulatory protein kinases in VSM as potential targets for antihypertensive therapy, as even small reductions of BP can have clinically significant effects.

Several potential kinases have been identified that target RLC₂₀, including ROCK (Amano et al., 1996), the ROCK-related citron kinase (Yamashiro et al., 2003), the zipper interacting protein kinase (ZIPK) (Murata-Hori et al., 1999; Niiro and Ikebe, 2001), integrin-linked kinase (ILK) (Deng et al., 2001; Walsh, 2011), the Inhibitor κΒ kinase 2 (IKK2) (Ying et al., 2013) and the PIM3 (Proviral Integration site for Moloney murine leukemia virus) kinase (Carlson et al., 2018). These kinases have been shown to phosphorylate both Ser19 and the adjacent Thr18 in vitro. However, the specific physiological implications of in vitro studies are not fully understood. There is evidence that PIM/ZIPK contribute to contraction and maintenance of vascular tone, and that inhibitors of these kinases can reduce BP in mouse models (Carlson et al., 2018). Further, IKK2 deficient mice have been found to have decreased aortic contractile responses and reduced responses to hypertensive vasoconstrictors, suggesting a potential role in regulating vascular function and BP (Ying et al., 2013).

The p90 ribosomal S6 kinase isoform 2 (RSK2), encoded by the gene RPS6KA3, was another kinase observed to phosphorylate MLCK in vitro on Ser19 (Suizu et al., 2000). We recently demonstrated that RSK2 contributes to basal vascular tone, myogenic vasoconstriction of resistance arteries and blood pressure (BP) (Artamonov et al., 2013a; Artamonov et al., 2018). Our research revealed that this is mediated by direct phosphorylation of RLC₂₀ as well as phosphorylation of the Na⁺/H⁺ exchanger, NHE1, which leads to an alkalinization of the SM cytosol and increased cytosolic [Ca²⁺], activating MLCK and/or other Ca²⁺-dependent kinase(s) (Artamonov et al., 2018). RSK kinases (four isoforms) are atypical in that they have two catalytic domains: the functionally important N-terminal catalytic domain (NTKD) and the regulatory C-terminal (CTKD). The activation of RSK2 in cells is triggered by the phosphorylation of CTKD by ERK1/2 kinases on several residues, including Ser386, which creates a docking site for 3-phosphoinositide-dependent protein kinase 1 (PDK1). The latter phosphorylates and activates NTKD on Ser227 (Anjum and Blenis, 2008; Pearce et al., 2010; Lara et al., 2013). We observed that increased intraluminal pressure in resistance vessels to induce myogenic vasoconstriction results in phosphorylation of Ser227 of endogenous RSK2; moreover, vasoconstriction and Ser227 phosphorylation as well as RLC₂₀ phosphorylation are blocked by RSK inhibitors BI-D1870 and LJH685 (Artamonov et al., 2013a; Artamonov et al., 2018). RSK2 signaling accounts for approximately 25% of the maximal myogenic constriction in normal mouse resistance arteries, implicating RSK2 as a significant player in vasoconstriction under physiological conditions. Accordingly, BP was significantly lower and the myogenic response suppressed in mice with global RSK2 knockout (Artamonov et al., 2018). Data suggest, RSK2 may account for the contractility observed in the absence of MLCK and could be a viable drug target for hypertension.

To understand the role of RSK2 in SM function in a rigorous manner, it was necessary to conduct experiments utilizing a mouse model with complete mylk1 knockout. Previously, we reported generation of a global mylk1 knockout in mice, by disrupting promotor sequences located within intron 27 (Somlyo et al., 2004). Although the embryos developed to full size, they died within 1–5 h after birth. This KO mouse will subsequently be referred to as mylk1 ^−/−. The expression of all three mylk1 products in these mice was abolished, as evidenced by Western blot and polymerase chain reaction analysis. Unfortunately, at the time, we did not pursue further experimental characterization of the SM tissues from these embryos; we completed the work now and are reporting the full results. Further, we gained insights into the mechanisms responsible for contractions and RLC₂₀ phosphorylation that occur in the absence of MLCK, including importantly the role of RSK2. Our data corroborate that RSK2 mediates these contractions. Additionally, we present compelling evidence that the apparent Ca²⁺-dependence of RSK2 is mediated by the upstream activity of the Ca²⁺ activated tyrosine kinase, Pyk2, which leads to the activatory phosphorylation of PDK1 and/or ERK1/2, with consequent upregulation of RSK2. This is consistent with previous findings about the physiological role of Pyk2 in SM. (Eguchi et al., 1999; Taniyama et al., 2003). We also report that similar to MLCK, RSK2 binds to actin and to MLCK forming a signaling hub with PDK1 and ERK1/2 on the actin filament, ideally positioned to interact with the myosin RLC₂₀.

Our findings provide further support of an important role for RSK2 signaling in vascular SM contractility, and provide rationale for the observed contractility of SM in tissues with mylk1 knockout (mylk1 ^−/− ).

Generation of MLCK/telokin KO mouse: While generating the telokin-null mouse by homologous recombination (Khromov et al., 2006), we found that insertion of the full-length targeted construct into this locus, including the loxP flanked neomycin selection cassette, resulted in complete disruption of the mylk1 gene expression in the absence of Cre recombination (Somlyo et al., 2004). The mice died shortly after birth. At E14.5 the litters (n = 125) showed close to the expected Mendelian inheritance ratio of 1.5^−/−:4.8^+/−:1^+/+ with a normal average litter size of 8. Embryonic mylk1 ^−/− aortic SM cells showed a complete absence of 220 and 130 kDa isoforms of MLCK (Figure 1A left panel) as evidenced by Western blotting. 17 kDa telokin is also absent in mylk1 ^−/− aortic SM cells (Supplementary Figure S1B). In tissues the long form of MLCK is not expressed in either E18.5 embryonic WT or mylk1 ^+/− bladder with lung serving as a positive control. The short 130 kDa isoform of MLCK, is present as expected. Telokin, a non-kinase SM-specific 17 kDa protein, identical to the C-terminus of MLCK is also absent in the mylk1 ^−/− . Telokin is phosphorylated by PKA/PKG (Wu et al., 1996; Wu et al., 1998; Khromov et al., 2006). When phosphorylated telokin binds to the phosphorylated myosin phosphatase facilitating its binding to phosphorylated myosin and promoting myosin dephosphorylation and relaxation (Khromov et al., 2012). The lack of telokin in the mylk1 ^−/− embryos and SM cells would tend to increase RLC₂₀ phosphorylation and basal force levels, yet SM containing tissues appeared dilated (Figure 1) and calyculinA forces were not different in WT and mylk1 ^−/− SM. Furthermore, resting basal cyclic nucleotide-induced phosphorylation of telokin should be low (Renna et al., 2013). Telokin expression is also fivefold lower in vascular SM, used as a source of cultured embryonic SM cells, and for some contractility studies, compared to gut smooth muscle (Khromov et al., 2006).

Pathophysiology of vascular smooth muscle (VSM) constitutes a critical element of the etiology of hypertension. Specifically, increased sustained vascular contraction, i.e., higher tone, leads to increased vascular resistance due to reduced vascular diameter and arterial remodeling (Renna et al., 2013). Hypertension is a major risk factor for heart failure, myocardial infarction, stroke, vascular dementia, and other diseases, but the current pharmacotherapiesrelying primarily on diuretics, β-blockers, Ca²⁺-channel blockers and renin-angiotensin pathway inhibitors—have limited success rates, leaving behind a significant number of patients refractory to treatments. It is not surprising that molecular mechanisms governing VSM contraction are therefore actively studied in search of alternative drug targets. Most relevant cellular signaling pathways are regulated by protein phosphorylation, catalyzed by specific kinases. The latter are attractive drug targets, and many kinase drugs have been approved in the last two decades for clinical use, especially to treat cancers (Cohen, 2002; Attwood et al., 2021). And the field of design of kinase inhibitors for use in the clinic is highly advanced, with 68 approved by FDA as of 2022 (Roskoski, 2022). Therefore, kinases involved in VSM regulation might potentially offer new avenues for the treatment of hypertension.

As described in the Introduction, recent studies showed that RLC₂₀ is a substrate for phosphorylation by protein kinases other than MLCK1. The fact that complete global deletion of the mylk1 gene does not prevent formation of embryos—even though they die after birth (Somlyo et al., 2004)—provides compelling evidence that auxiliary kinases activating SM myosin are physiologically important in SM, and capable for compensating for lack of MLCK1. However, auxiliary kinases could not substitute for all MLCK functions as SM containing tissues such as aorta, bladder, trachea and esophagus from mylk1 ^−/− mice were markedly dilated. Dilated irregular subepicardial coronary vessels were observed from mylk1 ^−/− mice. Here, we expand the characterization of the mylk1 ^−/− global knockout, and demonstrate that SM tissues including bladder, fundus and blood vessels, as well as cultured SM cells, retain the ability to contract and migrate through the normal pathway, as evidenced by increased RLC₂₀ phosphorylation in response to agonists (Figure 3). Importantly, addition of the MLCP inhibitor, calyculin A, to mylk1 ^−/− arteries and bladder in the absence of Ca²⁺ induces contractions of similar magnitude to those seen in normal arteries of similar size (Figure 2A). Therefore, under these conditions, the auxiliary RLC₂₀ kinase is able to induce the full force potential of SM, and indicates that the contractile apparatus is intact. Based on the calyculinA contractions, this kinase appears to be Ca²⁺-independent and operating in mylk1 ^−/− tissues, in the presence of intact RhoA/ROCK pathway (Scheme, Figure 6C), which is also fully functional because embryonic SM tissues contract to GTPγS stimulation at constant [Ca²⁺] and inhibition of ROCK relaxes the GTPγS contractile responses (Figure 2D).

The percent relaxation due to the increased phosphatase activity, upon inhibition of ROCK, was significantly greater in the mylk1 ^−/− SM as the magnitude of contractile force depends on the ratio of kinase to phosphatase activity. RSK2 kinase activity is less and slower than MLCK, altering this ratio and allowing a preponderance of ROCK activated phosphatase activity. Likewise, the smaller and slower initial phasic high [K⁺] contraction in the mylk1 ^−/− compared to the mylk1 ^+/+ arteries reflects the depolarization induced rapid Ca²⁺ transient that activates Ca²⁺/CaM/MLCK present in the mylk1 ^+/+ but lacking in the mylk1 ^−/− arteries (Figures 2A, B).

The challenge now is to identify the most relevant protein kinase in VSM that might constitute a good target for therapy. We recently presented strong evidence supporting the notion that this role is played by the p90 ribosomal S6 kinase (RSK2), coded by the RPS6KA3 gene, which has been hitherto recognized primarily for its role in brain development [RPS6KA3 mutations cause the Coffin-Lowry developmental syndrome (Hanauer and Young, 2002; Jacquot et al., 2002)] and cancer progression [it is a recognized target for cancer therapy, particularly the triple-negative breast cancer (Huynh et al., 2020)]. Specifically, previously we used SM tissues from global RPS6KA3 ^−/− mice, which are viable, to demonstrate the impact of deletion of RSK2 from SM (Artamonov et al., 2018). In the present, complementary study, we further explored the role of RSK2 in the SM tissues from mylk1 ^−/− embryos.

Arguably the most direct and compelling confirmation of our hypothesis in the present study is the fact that specific RSK2 inhibitors, Bi-D1870 and LJH685, inhibited carbachol-induced contractility in bladder of the mylk1 ^−/− embryos (Figure 4), as well as RLC₂₀ phosphorylation on Ser (Carlson et al., 2018) in mylk1 ^−/− SM cells stimulated with either LPA or U46619 (Figure 3). Therefore, in the absence of MLCK, contractility appears to be regulated primarily by the activities of RSK2 and the RhoA/Rock signaling pathways (Scheme, Figure 6C). The low, residual contractility and RLC₂₀ phosphorylation still observed in the presence of the RSK2 inhibitors may be due to incomplete RSK2 inhibition or to other kinases such as RSK1, integrin-linked kinase (Deng et al., 2001; Wilson et al., 2005), PIM/ZIPK (Carlson et al., 2018), or IKK2 (Ying et al., 2013). Nevertheless, our results support the notion that RSK2 is the long sought primary auxiliary contributor to SM contractility, independent of MLCK activity.

In normal SM cells, stimulation activates both MLCK and RSK2. In this case, as we have shown previously (Artamonov et al., 2018). RSK2 can both directly phosphorylate RLC₂₀ and also activate the Na⁺/H⁺ exchanger NHE-1 resulting in an alkalinization of the cytosol, an increase in [Ca²⁺] by a yet to be identified mechanism, and amplification of MLCK activity (Scheme, Figure 6C). When these pathways dominate over MLCP activity, the net result is constriction, as in the case of myogenic vasoconstriction induced by agonist or intraluminal pressure, or in maintenance of basal tone in the resting state. It is however, of importance to note that not all three pathways are activated equally by different stimuli, or have the same time course (Artamonov et al., 2013b). For example, thromboxane analogue U46619 is a specific potent activator of the RhoA signaling. On the other hand, the onset of depolarization with high [K⁺] is dominated by Ca²⁺/CaM/MLCK signaling as seen in Figure 2A when comparing initial high [K⁺] responses in the arteries from the normal and mylk1 ^−/− mice. RSK2 signaling, in turn, contributes ∼25% of the myogenic vasoconstriction response in resistance arteries, and contributes to resting blood pressure control (Artamonov et al., 2018). These multiple pathways provide both redundancy but also specialization depending on the stimuli, resulting in phasic or tonic type contractions.

Although RSK2 activity appears to be independent of MLCK, in normal SM cells MLCK immunoprecipitates with RSK2 and actin and this association increased with stimulation. The co-localization was confirmed in PLA assays, showing they are <40 nm apart. MLCK has an actin-binding site in its N-terminus with its C-terminal kinase domain poised to reach across to interact with, and phosphorylate myosin heads to initiate myosin cross bridge cycling (Lin et al., 1999). We have previously reported that a fraction of RSK2 immunoprecipitates with actin filaments (Artamonov et al., 2018), while a soluble fraction is free to phosphorylate other substrates, such as the membrane associated Na⁺/H⁺ exchanger NHE-1. Interestingly, the two upstream activators of RSK2—PDK1 and ERK1/2— were also brought down with the immunoprecipitated complex of RSK2/MLCK and actin, suggesting a signaling hub on the actin filament poised to phosphorylate and activate neighboring myosin filaments. Few puncta were observed in the (−) serum PLA assay unlike the (−) serum immunoprecipitation assay where MLCK pulls down actin plus RSK2, ERK1/2 and PDK1. (+) serum increased the immunoprecipitation of RSK2 and phospho-RSK2. The immunoprecipitation assay requires homogenization of the sample which may have partially activated the associations unlike the PLA assay carried out on intact cells. In this case, RSK2, possibly complexed to ERK1/2 and PDK1 may be bound to actin and not directly to MLCK, so not detected in the unstimulated SM cells in the PLA assay. It remains to be determined, if the proximity of RSK2 to MLCK alters its activity and vice versa.

An intriguing question also remains as to how low (∼1 μM) concentrations of kinases tethered to actin filaments can phosphorylate ∼100 μM myosin heads. One reported possibility is that MLCK diffuses along actin filaments and enhances the rate of phosphorylation of SM myosin, as described by others (Hong et al., 2015). In that study, single molecules of fluorescent quantum dot labeled MLCK moved along actin filaments and preferentially located to areas in which myosin was not yet phosphorylated. The motion occurred only if the acto-myosin and MLCK-myosin interactions were weak. If the RSK2 associated with the MLCK/actin complex also moves along actin, this could similarly phosphorylate myosin heads provided that the RSK2 catalytic N-terminal domain can physically reach the myosin heads. Otherwise, the activated MLCK-associated RSK2 may regulate the activity of its MLCK partner.

One of our major concerns was that the results concerning [Ca²⁺] dependence of the auxiliary pathway were apparently contradictory. While the RSK2-mediated contractile force developed in mylk1 ^−/− SM in response to calyculin A was independent of Ca²⁺ (pCa > 8), the permeabilized mylk1 ^−/− SM contracted in response to increased [Ca²⁺] (Figure 2C), with maximal force at pCa4.5 being ∼3 fold lower in the mylk1 ^−/− bladder. As the magnitude of contraction and RLC₂₀ phosphorylation reflects the ratio of kinase:phosphatase activity and assuming phosphatase activity is constant, the MLCK activity present in the WT accounts for its greater force at pCa 4.5. We previously reported that the pCa-force relationship in α-toxin permeabilized rabbit arteries was right-shifted and the maximal [Ca²⁺] response was inhibited by as much as ∼ 60% by either the RSK2 inhibitor BI-D1870 or the PDK1 inhibitor GSK2334470. These data suggest Ca²⁺-dependence of RSK2 and PDK1. AngII stimulation of SM cells from human resistance arteries has been shown to increase intracellular [Ca²⁺] and to induce a cytosolic alkalinization, which leads to increased activities of ERK(1/2) and several tyrosine kinases (He et al., 1998). These changes in [Ca²⁺] and alkalinization are consistent with AngII activation of RSK2 signaling (Artamonov et al., 2018) and the tyrosine kinase activity suggest a role for another kinase. The non-receptor proline-rich tyrosine kinase Pyk2 is Ca²⁺-dependent and phosphorylates PDK1 in AngII stimulated vascular SM (Taniyama et al., 2003). Pyk2 has also been shown to play a role in contraction of arterial SM (Mita et al., 2013; Mills et al., 2015). Knockdown of PDK or Pyk2 inhibits LPA-induced phosphorylation of RSK2 and NHE3 activity in immortalized human colorectal adenocarcinoma cells (No et al., 2015). Similarily, in our studies, inhibition of Pyk2 inhibits AngII phosphorylation of Pyk2, PDK1 and RSK2 (Figure 6A). Activation of the RSK2 pathway, resulting in activation of NHE-1 and an increase in Ca²⁺ events (Taniyama et al., 2003), may also contribute to Pyk2 activation. Whether this increase in Ca²⁺ and/or the Ca²⁺ influx and release from stores provides the source for activation of Pyk2 remains to be determined. Using a highly selective inhibitor of Pyk2, PF4618433 (Han et al., 2009), we have found that AngII phosphorylates Pyk2 in aortic SM cells. PF4618433 inhibits both phosphorylation of Pyk2 and its downstream target PDK1 as well as its downstream target RSK2 phosphorylation. Altogether, these findings are consistent with Pyk2 being upstream of PDK1 and RSK2 and accounting for the observed Ca²⁺-dependent component of RSK2 activity (scheme, Figure 6C). Based on the magnitude of this Ca²⁺-dependent component in the absence of MLCK (Figure 2C), it accounts for approximately 25% of the maximal contractile force in the WT. Maximal force in response to CalyculinA inhibition of MLCP was equivalent in WT and mylk1 ^−/− smooth muscles (Figure 2A).The Ca²⁺-independent component in the mylk1 ^−/− SM reflects the RSK2 direct phosphorylation of myosin RLC₂₀ and RSK2 activation of NHE-1 leading to increased Ca²⁺ events.

The limited tissue size, fragility and availability from the E18.5 embryos did not allow us to make a rigorous comparison of the impact of deletion of MLCK on the contractile properties of different types of SM. The histology showed that bladder, bronchi, trachea and blood vessels were dilated in the mylk1 ^−/− embyros compared to WT littermates. We were able to compare contractile responses to high K⁺ treatment in bladder and artery (Figure 2B). In both cases the t_1/2 was significantly slower in the mylk1 ^−/− compared to WT SM but the bladder was faster than the artery, in both the presence or absence of MLCK. The tonic component of the high K⁺ stimuli predominated in the bladder (Figure 4A). All SM tissues and cells lacking mylk1 ^−/− displayed evidence for an auxiliary kinase(s) other than MLCK that supports contractility, RLC₂₀ phosphorylation and RSK2 signaling pathways. While determining the heterogeneity of different embryonic SM tissues responses was not feasible, all experiments supported our hypothesis that RSK2 kinase plays a significant role in SM contractility. The magnitude in different SMs remains to be determined. It is∼25% of maximal force in mouse resistance mesesenteric arteries (Artamonov et al., 2018).

In conclusion, we present compelling evidence that RSK2 largely accounts for the contractile and phosphorylated RLC₂₀ myosin observed in response to stimuli in SM from the mylk1 ^−/− embryos. These embryos have also allowed for the determination of the contributions of the RSK2 signaling to SM contractility in the complete absence of the canonical Ca²⁺/CaM/MLCK signaling. These findings support our previous studies showing the physiological importance of RSK2 signaling in the regulation of SM contractility and resting blood pressure. RSK2 contributes ∼25% of maximal force generated in response to increased intraluminal pressure in normal resistance arteries that regulate blood pressure and basal tone (Artamonov et al., 2018). Altogether, we propose that there are three major signaling pathways, driven by MLCK, RSK2 and RhoA/ROCK that regulate RLC₂₀ phosphorylation and force in smooth muscle (Scheme, Figure 6C). The magnitude of their contributions depends on the stimuli and the smooth muscle tissue.