Loop 2 of<i>Limulus</i>Myosin III Is Phosphorylated by Protein Kinase A and Autophosphorylation

Kempler, K; Tóth, Judit; Yamashita, Roxanne A.; Mapel, G.M.; Robinson, Kimberly; Cardasis, Helene L.; Stevens, Stanley M.; Sellers, James R.; Battelle, Barbara‐Anne

doi:10.1021/bi062112u

Cited by 26 publications

(49 citation statements)

References 72 publications

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“…Our conclusion was that Myo9b reversibly switches between a high affinity F-actin-binding state and a state that is not binding to F-actin even in the time scale of the cosedimentation experiments (6). This is reminiscent of Limulus myosin III preparations that also contained mixtures of two populations, one competent and one incompetent to bind F-actin (34). These observations resemble also findings reported for the heterodimeric microtubule-dependent motor Kar3/Vik1 containing catalytic Kar3 and the nonmotor protein Vik1.…”

Section: Discussionsupporting

confidence: 81%

Functional Role of the Extended Loop 2 in the Myosin 9b Head for Binding F-actin

Struchholz

Elfrink

Pieper

et al. 2009

Journal of Biological Chemistry

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The mammalian class IX myosin Myo9b can move considerable distances along actin filaments before it dissociates. This is remarkable, because it is single headed and because the ratelimiting step in its ATPase cycle is ATP hydrolysis. Thus, it spends most of its cycling time in the ATP-bound state that has a weak affinity for F-actin in other myosins. It has been speculated that the very extended loop 2 in the Myo9b head domain comprises an additional actin-binding site that prevents it from dissociation in the weak binding states. Here we show that two regions in the loop 2 determine the F-actin concentrations needed to maximally activate the steady-state ATPase activity. Together these two regions regulate the amount capable of binding F-actin and the affinity of the nucleotide-free state. The isolated loop 2 behaved like an entropic spring and bound stoichiometrically and with high affinity to F-actin. Subfragment 1 from skeletal muscle myosin II bound to F-actin simultaneously with the isolated loop 2 of Myo9b and could not displace it. Furthermore, the present results imply also a regulatory role for the tail region. Taken together, the results demonstrate that the extended loop 2 in Myo9b binds F-actin and influences the binding of the conventional stereo-specific actin-binding site.Myosin 9b (Myo9b, myr 5) 2 has been reported to move processively along actin filaments, i.e. upon binding to an actin filament it takes multiple consecutive steps before it dissociates (1-3). This is remarkable, because Myo9b is a single-headed myosin. Other myosins that move processively are two-headed and coordinate movement between the two heads (4, 5). Myo9b is also unique in that ATP hydrolysis is the rate-limiting step in the ATPase cycle (6, 7). This means that Myo9b spends a considerable amount of its cycling time in the ATP-bound state that represents a typically weak actin affinity state. However, Myo9b in the ATP-bound state binds with a relatively high affinity to F-actin (6, 7). Nevertheless, kinetic data do not unequivocally support processive movement.It has been speculated that the exceptionally long insertion at the position of loop 2 in the myosin head tethers Myo9b to F-actin and prevents it from diffusing away. The loop 2 in myosins is a surface loop that has been implicated in the initial weak electrostatic interaction with F-actin. In the processive myosin Va this loop is a little longer and more positively charged than in the non-processive class II myosins. An increase in the net positive charge of loop 2 increased the affinity of myosin Va for F-actin in all nucleotide states, whereas a decrease in its net positive charge reduced the affinity (8). Similar findings were also obtained with naturally occurring splice variants of the non-processive myosin V from Drosophila melanogaster and by modifying the loop 2 in class II myosins (8 -12). The processive run length of myosin Va varied with the affinity for F-actin in the weak binding states (13,14). A higher net positive charge of loop 2 increased the pro...

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Section: Discussionsupporting

confidence: 81%

Functional Role of the Extended Loop 2 in the Myosin 9b Head for Binding F-actin

Struchholz

Elfrink

Pieper

et al. 2009

Journal of Biological Chemistry

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“…Kinase activity is conferred by an N-terminal kinase domain prior to the myosin motor domain which is dependent on autophosphorylation. Phosphorylation of residues in an actin-binding surface loop of the myosin motor domain of human myosin-3a decreases the actin affinity under steady-state conditions > 100 fold along with the duty ratio of the motor [79, 80]. Deletion of the kinase domain increases the ATPase activity, actin affinity and duty ratio of human myosin-3a, implying that autophosphorylation reduces motor activity [81].…”

Section: Regulation By Phosphorylationmentioning

confidence: 99%

Various Themes of Myosin Regulation

Heissler

Sellers

2016

Journal of Molecular Biology

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114

127

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Members of the myosin superfamily are actin-based molecular motors that are indispensable for cellular homeostasis. The vast functional and structural diversity of myosins accounts for the variety and complexity of the underlying allosteric regulatory mechanisms that determine the activation or inhibition of myosin motor activity and enable precise timing and spatial aspects of myosin function at the cellular level. This review focuses on the molecular basis of posttranslational regulation of eukaryotic myosins from different classes across species by allosteric intrinsic and extrinsic effectors. First, we highlight the impact of heavy and light chain phosphorylation. Second, we outline intramolecular regulatory mechanisms such as autoinhibition and subsequent activation. Third, we discuss diverse extramolecular allosteric mechanisms ranging from actin-linked regulatory mechanisms to myosin:cargo interactions. At last, we briefly outline the allosteric regulation of myosins with synthetic compounds.

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“…NINAC motor activity has not been demonstrated; therefore, it has been proposed that G q α translocation involves reversible associations of G q α with the cytoplasmic and rhabdomeral isoforms of NINAC (Montell, 2012). Limulus eyes express a single homolog of NINAC that is distributed throughout the photoreceptor, concentrated at the rhabdoms (Battelle et al, 2001), and is a major target for phosphorylation by the circadian clock via activation of cAMP-dependent protein kinase (Edwards and Battelle, 1987;Battelle et al, 1998;Kempler et al, 2007;Cardasis et al, 2007). Limulus myosin III is not a molecular motor ; however, based on the findings in Drosophila and the results presented here, it is interesting to speculate that the clock-and cAMP-dependent phosphorylation of Limulus myosin III plays a role in the clock-dependent translocation of G q α to the rhabdom.…”

Section: The Clock Regulates the Increase In Rhg Q α Levels In The Darkmentioning

confidence: 99%

Opsin1-2, Gqα and arrestin levels at Limulus rhabdoms are controlled by diurnal light and a circadian clock

Battelle

Kempler

Parker

et al. 2013

Journal of Experimental Biology

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in white-eyed Drosophila maintained under artificial light. Here we tested whether protein levels at rhabdoms change significantly in the highly pigmented lateral eyes of wild-caught Limulus polyphemus maintained in natural diurnal illumination and whether these changes are under circadian control. We found that rhabdomeral levels of opsins (Ops1-2), the G protein activated by rhodopsin (G q α) and arrestin change significantly from day to night and that nighttime levels of each protein at rhabdoms are significantly influenced by signals from the animalʼs central circadian clock. Clock input at night increases Ops1-2 and G q α and decreases arrestin levels at rhabdoms. Clock input is also required for a rapid decrease in rhabdomeral Ops1-2 beginning at sunrise. We found further that dark adaptation during the day and the night are not equivalent. During daytime dark adaptation, when clock input is silent, the increase of Ops1-2 at rhabdoms is small and G q α levels do not increase. However, increases in Ops1-2 and G q α at rhabdoms are enhanced during daytime dark adaptation by treatments that elevate cAMP in photoreceptors, suggesting that the clock influences dark-adaptive increases in Ops1-2 and G q α at Limulus rhabdoms by activating cAMP-dependent processes. The circadian regulation of Ops1-2 and G q α levels at rhabdoms probably has a dual role: to increase retinal sensitivity at night and to protect photoreceptors from light damage during the day.

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Loop 2 ofLimulusMyosin III Is Phosphorylated by Protein Kinase A and Autophosphorylation

Cited by 26 publications

References 72 publications

Functional Role of the Extended Loop 2 in the Myosin 9b Head for Binding F-actin

Functional Role of the Extended Loop 2 in the Myosin 9b Head for Binding F-actin

Various Themes of Myosin Regulation

Opsin1-2, Gqα and arrestin levels at Limulus rhabdoms are controlled by diurnal light and a circadian clock

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