SHARPIN serine 146 phosphorylation mediates ARP2/3 interaction, cancer cell invasion and metastasis

The adaptor protein SHARPIN is involved in a number of cellular processes and promotes cancer progression and metastasis. However, how the choice between different functions of SHARPIN is post-translationally regulated is unclear. Here we have characterized SHARPIN phosphorylation by mass spectrometry and in vitro kinase assay. Focusing on two uncharacterized phosphorylation sites, serine 131 and 146, in the unstructured linker region of SHARPIN, we demonstrate their role in SHARPIN-ARP2/3 complex interaction, whereas they play no role in integrin inhibition or LUBAC activation. Consistent with its novel role in ARP2/3 regulation, serine 146 (S146) phosphorylation of SHARPIN promoted lamellopodia formation. Notably, CRISPR-Cas9 mediated knockout of SHARPIN abrogated three-dimensional (3D) invasion of several cancer cell lines. The 3D invasion of cancer cells was rescued by overexpression of the wild-type SHARPIN, but not by SHARPIN S146A mutant, identifying S146 as an invasion promoting phosphorylation switch. Finally, we demonstrate that inhibition of phosphorylation at S146 significantly reduces the in vivo metastasis in the zebrafish model. Collectively, these results demonstrate that SHARPIN S146 phosphorylation constitutes a single functional determinant of cancer cell invasion both in vitro and in vivo.


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The primary cause for cancer-related deaths is metastasis (Steeg, 2016). Significant 39 improvements in cancer survival rates have been seen recently due to early diagnosis 40 and development of targeted therapies (Guan, 2015). Metastasis however, remains still 41 a hurdle that most cancer therapies are not able to overcome. Cancer metastasis involves 42 several critical steps: first, the cancer cell(s) needs to detach from the primary tumor. 43 Subsequentially, the detached cell needs to migrate into and through the surrounding 44 tissue, a step called invasion. Then the metastasizing cancer cell needs to travel through 45 the blood or lymph system, after which it needs to adhere to the secondary site, where it 46 once more needs to invade to reach its final destination (Fares, Fares et al., 2020). 47 Suppressing cancer metastasis by targeting any of these processes would be of an urgent 48 therapeutic need (Ganesh & Massagué, 2021). However, this would require a detailed 49 mechanistic understanding of how these processes are regulated, and consequently 50 identification of potential target mechanisms for anti-metastatic therapies.  were grown in DMEM with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin.

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PC3 cells were grown in RPMI with 10% FBS, 1% penicillin-streptomycin and 1% L-124 glutamine. All cell lines were regularly tested for contaminations and were from American  were then cut out for mass spectrometry. Protein samples were then digested by trypsin. HeLa cella were seeded on to a 6 well plate. Next day cells were transfected with Control 168 or Sharpin siRNA. Following day these cells were transfected with GFP control, GFP 169 SHARPIN WT, GFP SHARPIN S131A or GFP SHARPIN S146A. The following day cells with 10% FBS was placed on top of the matrix in the inserts providing a serum gradient.

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Cells were fixed after 24-48 hours of seeding. 4% PFA was used to fix cells for 2 hours.

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Cell permeabilization was done using 0.5% Triton-X 100 at room temperature for 30 min.  analyzed by affinity purification coupled with mass spectrometry (AP-MS) (Fig. 1B). The 277 MS analysis revealed 7 SHARPIN phospho-sites; out of which serine 131 (S131), S146, 278 S165, threonine 309 (T309), and S312 were overlapping with IVK sites (Table 1) 289 We selected S131 and S146 for further functional analysis due to their presence in both 290 the IVK and in cellulo MS analysis (Table 1), as well as their clustering to an unstructured 291 linker region of SHARPIN the function of which is yet unknown (Fig. 1C). To investigate 292 the functional role of S131 and S146 phosphorylation, we created alanine mutants of  significantly inhibited integrin activity in these SHARPIN depleted cells (Fig. 2A). 305 However, as both phospho-mutants also significantly inhibited integrin activity, we 306 conclude that these phosphorylation sites are not relevant for the ability of SHARPIN to 307 inhibit integrins (Fig 2A). To analyze the effect of S131 and S146 phosphorylation sites on LUBAC activation, we 319 used the NF-KB activity luciferase reporter assay in HeLa cells. As expected, a loss of 320 SHARPIN significantly reduced NF-kB activity (Fig S1D), while overexpression of GFP 321 SHARPIN-WT increased NF-kB activity (Fig. 2B). Consistent with a previous report (De 322 Franceschi et al., 2015), the structural mutant L276A was incompetent to promote NF-kB 323 activity (Fig. 2B). Notably, the S131A and S146A mutants were indistinguishable from 324 SHARPIN-WT in their capacity to promote NF-kB activity (Fig. 2B), demonstrating that,

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As expected, no FRET signal was observed in cells with over-expression of GFP-332 SHARPIN-WT alone, whereas its co-expression with ARP3RFP resulted in a clear FRET 333 signal (Fig. 2D). Interestingly, FRET activity in cells expressing GFP-SHARPIN S131A or 334 S146A mutants was significantly lower as compared to the GFP-SHARPIN WT 335 expressing cells, and the activity with S146A was indistinguishable from the structural  These data demonstrate that the studied SHARPIN phosphorylation sites are not involved 339 in the integrin inhibition, or LUBAC regulation, but they significantly contribute to 340 SHARPIN-ARP3 interaction. Out of these two mutations, S146A had clearly stronger 341 effect on ARP3 interaction, and it was thus selected for the further functional validation.  (Fig. 3A). In a rescue experiment where 352 SHARPIN-silenced cells were transfected with either GFP-only, GFP SHARPIN-WT, 353 S146A, or V240A/L242A double mutant as a negative control (Khan et al., 2017), only the 354 SHARPIN-WT was able to rescue the lamellipodium formation in (Fig 3B). These data 355 are consistent with the FRET data (Fig. 2D), and indicate that phosphorylation of S146 is 356 required for SHARPIN-mediated ARP2/3 activation. As S146 was found phosphorylated in the unperturbed cancer cells (Table I), we assumed 369 that overexpression of phosphomimic glutamate mutant of S146 (S146E) would not 370 impact ARP2/3 interaction or lamellipodium formation by SHARPIN. Use of the S146E 371 mutant would also be an important control that the impaired lamellipodia formation by 372 S146A mutant was truly due to lack of phosphorylation, and not due to structural impact 373 of any random mutation. Importantly, although GFP-SHARPIN S146E showed slightly 374 reduced binding to ARP3-RFP (Fig. 3C), its overexpression resulted in comparable 375 rescue of lamellipodia formation as compared to GFP-SHARPIN WT expressing cells 376 (Fig. 3D). Thereby we conclude that the lack of lamellipodia rescue with the S146A mutant 377 was due to impairment of phosphorylation at S146.  Single phosphorylation site S146 on SHARPIN determines cancer cell 417 invasiveness. 418 The results above demonstrate that SHARPIN S146 phosphorylation promotes 419 lamellipodia formation (Fig. 3), which is a known requirement for cancer cells invasion, 420 and that SHARPIN is required for 3D invasion across cancer cell lines (Fig. 4). To    (Table 1). Whereas previous study 479 demonstrated the functional role of S165 phosphorylation on SHARPIN-mediated LUBAC 480 regulation, the role of the other SHARPIN phosphorylation sites has not been studied as 481 yet. Here we focused on functional analysis of S131 and S146 phosphorylation as these 482 sites were observed phosphorylated both on mass spectrometry and the IVK data ( Fig.   483 1C and Table 1).

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Prior this study, SHARPIN was known to promote lamellipodium formation through 486 interaction with the ARP2/3 complex, and it was further demonstrated that this function 487 was independent of its LUBAC-and integrin related roles (Khan et al., 2017). Here we 488 demonstrate role for S146 and S131 phosphorylations on SHARPIN-ARP2/3 interaction, 489 and that mutations of these sites had no effect on the ability of SHARPIN to inhibit 490 integrins or on NF-KB activation. S146 phosphorylation of SHARPIN was further validated 491 to promote lamellipodia formation, but consistent with constitutive phosphorylation of 492 S146 based on mass spectrometry data, the phosphorylation mimicking mutation 493 (S146E) functioned as a WT. Furthermore, we demonstrate that phosphorylation of 494 SHARPIN at S146 translates into the ability of cancer cells to invade and to metastasize 495 in vivo.

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In summary, our data teases out a single phosphorylation event which is essential for