The Drosophila mutant turnip was initially isolated based on poor learning performance. turnip is dramatically reduced in protein
kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane
protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies.
Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map
genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently
turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. These
results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in
associative learning in Drosophila (Choi, 1991).
Activation of the Drosophila visual cascade is extremely rapid and results
in opening of the cation influx channels transient receptor potential (TRP)
and transient receptor potential-like (TRPL) within ~10-20 msec of
photostimulation of rhodopsin. The
G-protein-signaling cascade that leads to opening of the ion channels has
been extensively characterized and is known to involve the inositol
phospholipid-signaling system. Termination of the photoresponse, after
cessation of the light stimulus, is also rapid and is a Ca2+-regulated process; however, understanding of the mechanism by which Ca2+ contributes to termination of the photoresponse is quite
incomplete (Li, 1998 and references).
Several proteins have been identified that seem to mediate Ca2+-dependent termination of phototransduction.
These include the Ca2+-binding regulatory protein Calmodulin, which functions in both light adaptation and
termination of the light response.
The ninaC (neither inactivation nor afterpotential C) locus, which encodes two isoforms, p132 and p174,
each of which consists of a protein kinase domain fused to a myosin head domain, also functions in negative feedback regulation of the photoresponse. The two
NINAC proteins differ because of unique C-terminal ends. p174 is localized exclusively to the microvillar
portion of the photoreceptors, the rhabdomeres, and p132 is restricted to the cell bodies. Null mutations in ninaC cause defects in adaptation and response termination. These functions are caused by p174 because elimination of p174, but not p132, causes each of
these phenotypes. Because negative feedback regulation seems to be mediated by Ca2+, it is plausible that
p174 is regulated by Ca2+. However, p174 does not contain a known Ca2+-binding motif, such as an EF
hand or C2 domain, and there is no evidence that it binds Ca2+ directly. Thus, p174 seems to respond to the
light-dependent Ca2+ flux indirectly. One NINAC Ca2+ sensor is Calmodulin because NINAC binds to
Calmodulin and the NINAC-Calmodulin interaction is required for both adaptation and
termination. NINAC might also be regulated by
Ca2+-dependent phosphorylation because p174 contains multiple protein kinase C (PKC) consensus sites
including several in its unique C-terminal tail. Moreover, mutation of an eye-specific PKC (ePKC) causes
perturbations in adaptation and termination. The role of PKC in
negative feedback regulation may be more significant than that indicated by mutation of ePKC because a
second PKC, brain PKC (brPKC), is known to be enriched in the Drosophila retina and a third PKC,
PKC98F, is highly expressed in adult heads. Two retinal substrates for PKC have
been identified. These are the TRP cation influx channel and the PSD95, DLG, and
ZO-1 (PDZ)-containing protein inactivation, no afterpotential D (INAD), which binds
to most of the proteins that function in phototransduction and organizes a supramolecular signaling complex. However, the consequences of disrupting PKC phosphorylation of any
retinal substrate that functions in Drosophila vision have not been determined (Li, 1998 and references).
The current work shows that NINAC p174, which consists of a protein kinase domain joined to the head region of myosin heavy chain, is a phosphoprotein and is
phosphorylated in vitro by PKC. Mutation of either of two PKC sites in the p174 tail results in an unusual defect in deactivation that has not been detected
previously for other ninaC alleles or other loci. After cessation of the light stimulus, there appeared to be a transient reactivation of the visual cascade. This
phenotype suggests that a mechanism exists to prevent reactivation of the visual cascade and that p174 participates in this process. The termination mechanisms controlling Drosophila phototransduction seem to be more
complicated than previously envisioned. In addition to a requirement for NINAC in facilitating rapid
deactivation after cessation of the light stimulus, there is an additional requirement for this unconventional
myosin in preventing transient reactivation of the plasma membrane conductances. Because p174 also
functions in adaptation, it seems that NINAC has a central role in many aspects of negative feedback
regulation of the visual cascade. Recently, a homolog of NINAC has been identified in the mammalian retina
(D. Hillman, A. Dose, and B. Burnside, personal communication to Li, 1998). Thus, it is intriguing to speculate that
vertebrate NINAC also functions in negative feedback regulation and that an active mechanism may also
exist in mammalian photoreceptor cells to ensure stable termination of phototransduction (Li, 1998).
Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility. Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans. To investigate this
regulation in a complex eukaryote, the Drosophila myosin-II tail
expressed in Escherichia coli was purified and it was shown to be phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs. If the two serines are
mutagenized to alanine or aspartic acid, phosphorylation no longer occurs. Using
a 341 amino acid tail fragment, it has been shown that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized
polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, which is
encoded by the zipper gene, appears to be similar to rabbit nonmuscle
myosin-IIA. In vivo, transgenic flies were generated that expressed the various
myosin heavy chain variants in a zipper null or near-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations. These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way (Su, 2001).